Recombinant feline herpesvirus comprising a foreign DNA inserted into a region corresponding to a 3.0 kb EcoRI-SalI fragment of a feline herpesvirus genome

ABSTRACT

This invention provides a recombinant feline herpesvirus comprising a foreign DNA inserted into a feline herpesvirus genome, wherein the foreign DNA is inserted into a region of the genome which corresponds to the 3.0 kb EcoRI-SalI fragment within a SalI A fragment of the feline herpesvirus genome and is capable of being expressed in a host cell into which the virus is introduced. Further this invention provides a recombinant feline herpesvirus comprising a feline herpesvirus genome, wherein the feline herpesvirus genome contains a deletion in a SacII site within the 3.0 kb EcoRI-SalI fragment of the SalI A fragment of the feline herpesvirus genome. Lastly, this invention provides vaccines and methods of immunization of animals infected with feline herpesvirus.

Throughout this application various publications are referenced byArabic numerals in brackets. Full citations for these publications maybe found at the end of the specification immediately preceding theclaims. The disclosures of these publications are in their entiretyhereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

Herpesviruses contain 100,000 to 200,000 base pairs of DNA as theirgenetic material having a long unique segment and a short uniquesegment, the short unique segment bounded by an internal repeat sequenceand a terminal repeat sequence. Within a given genome, several regionshave been identified that are not essential for the replication of virusin cell culture. Modifications of some of these regions of the DNA havebeen known to lower the pathogenicity of the virus, i.e., to attenuatethe virus when it infects an animal species. For example, inactivationof the thymidine kinase gene of either human herpes simplex virus [1] orpseudorabies virus of swine [2] renders the human herpesvirus lesspathogenic in mice and the pseudorabies virus less pathogenic in swine.

Removal of specific regions of the repeat region of a human herpessimplex virus has been shown to render the virus less pathogenic [1, 3].Furthermore, a repeat region has been identified in Marek's diseasevirus that is associated with viral oncogenicity [4]. A region inherpesvirus saimiri has similarly been correlated with oncogenicity [5].Removal of a specific region of the repeat region renders pseudorabiesvirus less pathogenic (U.S. Pat. No. 4,877,737). A region inpseudorabies virus has been shown to be deleted in naturally-occurringvaccine strains [6]. These deletions are at least in part responsiblefor the lack of pathogenicity of these strains. Specific combinations ofglycoproteins gene deletions in herpes simplex virus render the herpessimplex virus less pathogenic in mice [see 37 for review]. While,combinations of glycoprotein gene deletions in pseudorabies virus renderthe pseudorabies virus less pathogenic in swine [see 38 for review].

Herpesviruses contain non-essential regions of DNA in various parts ofthe genome, and that modification of these regions can attenuate thevirus, leading to a non-pathogenic strain from which a vaccine may bederived. The degree of attenuation of the virus is important to theutility of the virus as a vaccine. Deletions which cause too muchattenuation of the virus will result in a vaccine that fails to elicitan adequate immune response. Although several examples of attenuatingdeletions are known, the appropriate combination of deletions for anyherpesvirus is not readily apparent.

Feline herpesvirus 1 (FHV) is the causative agent of feline viralrhinotracheitis, an acute upper respiratory disease in cats [7, 8].Serological studies indicate that 50 to 70% of adult cats havedetectable antibodies to the virus [9, 10]. Currently availableinactivated and attenuated live virus vaccines reduce disease but do notprevent infection by FHV [11].

The feline herpesvirus is a member of the family herpesviridae, whichare commonly known as the herpesviruses and a member of the subfamilyalphaherpesvirus with a group D genome [12]. The FHV genome is comprisedof approximately 134 kilobase (kb) pairs that is subdivided into a longunique segment of approximately 104 kb and a short unique segment ofapproximately 8 kb [13]. The unique short region is bounded by invertedrepeat sequences which are approximately 11 kb. Physical maps ofrestriction endonuclease sites of the FHV genome have been published[12, 13]. The thymidine kinase gene of FHV has been sequenced and an FHVvirus containing a deletion of the TK gene was isolated using a drugselection technique [14]. A feline herpesvirus having a deletion of theTK gene and an insertion of the feline leukemia virus (FeLV) envelope(env) and gag genes at the TK deletion site has been constructed [36].Feline herpesviruses containing an insertion in the ORF1 and ORF2 regionwithin the unique short [43], or a deletion in the gI and gE region ofthe unique short [44] have been described. A feline herpesviruscontaining an insertion in a region of the unique long downstream of theFHV gC gene has been constructed [40,45].

The feline herpesviruses in this invention are useful as vectors for thedelivery of vaccine antigens from microorganisms causing diseases inanimals other than cats or dogs and for the delivery of therapeuticagents. The therapeutic agent that is delivered by a viral vector of thepresent invention must be a biological molecule that is a by-product offeline herpesvirus replication. This limits the therapeutic agent in thefirst analysis to either DNA, RNA or protein. There are examples oftherapeutic agents from each of these classes of compounds in the formof antisense DNA, anti-sense RNA [18], ribozymes [19], suppressor tRNAs[20], interferon-inducing double stranded RNA and numerous examples ofprotein therapeutics, from hormones, e.g., insulin, to lymphokines,e.g., interferons and interleukins, to natural opiates. The discovery ofthese therapeutic agents and the elucidation of their structure andfunction does not necessarily allow one to use them in a viral vectordelivery system

SUMMARY OF THE INVENTION

This invention provides a recombinant feline herpesvirus comprising aforeign DNA inserted into a feline herpesvirus genome, wherein theforeign DNA is inserted into a region of the genome which corresponds toa 3.0 kb EcoRI-SalI fragment within a SalI A fragment of the felineherpesvirus genome and is capable of being expressed in a host cell intowhich the virus is introduced.

Further this invention provides a recombinant feline herpesviruscomprising a feline herpesvirus genome, wherein the feline herpesvirusgenome contains a deletion in a 3.0 kb EcoRI-SalI fragment of the SalI Afragment of the feline herpesvirus genome.

Lastly, this invention provides vaccines and methods of immunization ofanimals infected with feline herpesvirus.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Restriction endonuclease map of the feline herpesvirus genome.

FIG. 2: Restriction endonuclease map of the feline herpesvirus 3.0 kbEcoRI to SalI fragment within the SalI A genomic fragment. Three openreading frames have been identified in the 2968 base pair region (SEQ IDNO. 3),: UL 24 (SEQ ID NO. 4), UL 25 (SEQ ID NO. 5), UL 26 (SEQ ID NO.6).

FIGS. 3A-B: DNA insertion in Homology Vectors 855-30.A4. The diagramshows the orientation of DNA fragments assembled in plasmid 855-30.A4.The source of each fragment is described in the Materials and Methodssection. The origin of FHV DNA sequences is from SEQ ID NO: 3. Thesequences located at the junctions between fragments are shown (SEQ IDNOS: 13-16 respectively), including junction B (SEQ ID NO: 14), junctionE (SEQ ID NO: 15). The restriction endonuclease sites used to generateeach fragment as well as the synthetic DNA sequences that were used tojoin the fragments are described for each junction. The synthetic DNAsequences are underlined by a solid bar. The following convention isused: restriction endonuclease sites in brackets [] indicate theremnants of sites that were destroyed during construction. The DNAinsertion of the gX promoter-LacZ PRV gX poly A cassette (Fragments 2,3, and 4) is described in PCT International Publication WO 96/13575 andis incorporated herein by reference. The following abbreviations areused, feline herpesvirus (FHV), pseudorabies virus (PRV), glycoprotein X(gX), polyadenylation site (poly A), base pairs (BP), E. Colilacz-beta-galactosidase (lacZ).

FIGS. 4A-B: DNA insertion in Homology Vectors 855-30.F9. The diagramshows the orientation of DNA fragments assembled in plasmid 855-30.A4.The source of each The source of each fragment is described in theMaterials and Methods section. The origin of FHV DNA sequences is fromSEQ ID NO: 3. The sequences located at the junctions between fragmentsare shown (SEQ ID NOS: 17-20 respectively), including junction B (SEQ IDNO: 18), junction E (SEQ ID NO: 19). The restriction endonuclease sitesused to generate each fragment as well as the synthetic DNA sequencesthat were used to join the fragments are described for each junction.The synthetic DNA sequences are underlined by a solid bar. The followingconvention is used: restriction endonuclease sites in brackets []indicate the remnants of sites that were destroyed during construction.The DNA insertion of the gX promoter-lacZ PRV gX poly A cassette(Fragments 2, 3, and 4) is described in PCT International Publication WO96/13575 and is incorporated herein by reference. The followingabbreviations are used, feline herpesvirus (FHV), pseudorabies virus(PRV), glycoprotein X (gX), polyadenylation site (poly A), base pairs(BP), E. Coli lacZ-beta-galactosidase (lacZ).

FIG. 5: Junction region between feline herpesvirus glycoprotein Epromoter and foreign DNA (lacZ) in homology vector 846-88.B17 andrecombinant S-FHV-020 (SEQ ID NO: 23). The FHV gE. promoter is withingan approximately 1415 base pari Asp718 I to SmaI subfragment of the FHVSalI B genomic fragment.

DETAILED DESCRIPTION OF THE INVENTION

The present invention involves a recombinant feline herpesviruse usefulin the preparation of vaccines to protect cats from naturally-occuringinfectious feline herpesvirus. The present invention is also useful forexpression of a foreign DNA or genes from other pathogens for protectionagainst disease. Recombinant feline herpesvirus expressing a foreign DNAfrom avian or mammalian pathogens are useful as vaccines in avian ormammalian species including but not limited to chickens, turkeys, ducks,feline, canine, bovine, equine, and primate, including human.

This invention provides a recombinant feline herpesvirus comprising aforeign DNA inserted into a feline herpesvirus genome, wherein theforeign DNA is inserted into a region of the genome which corresponds toan approximately 3.0 kb EcoRI-SalI fragment within a SalI A fragment ofthe feline herpesvirus genome and is capable of being expressed in ahost cell into which the virus is introduced.

In one embodiment the foreign DNA is inserted within a UL25 gene withinthe 3.0 kb EcoRI-SalI fragment. In one embodiment the foreign DNA isinserted within a UL24 gene within the 3.0 kb EcoRI-SalI fragment. Inone embodiment the foreign DNA is inserted within a UL26 gene within the3.0 kb EcoRI-SalI fragment. In one embodiment the foreign DNA isinserted within a intergenic regions within the 3.0 kb EcoRI-SalIfragment. In another embodiment the foreign DNA is inserted within aNdeI site within the approximately 3.0 kb EcoRI-SalI fragment.

The “approximately 3.0 EcoRI-SalI fragment” means a fragment whichresults from the steps of: a) digesting a feline herpesvirus genome witha SalI restriction endonuclease, b) cloning the resulting fragments in aplasmid, and c) digesting the plasmid containing a SalI A fragment withan EcoRI and SalI restriction endonucleases. In one embodiment the“approximately 3.0 kb EcoRI-SalI fragment” is an EcoRI-SalI fragmentfrom a feline herpesvirus having a 2968 nucleotide acid sequence as setforth in SEQ. ID. NO: 3. It is contemplated by this invention that the“approximately 3.0 EcoRI-SalI fragment” is an EcoRI-SalI fragment whichis homologous to the EcoRI-SalI fragment of the 2968 kb EcoRI-SalIfragment from the NVSL strain.

For purposes of this invention, “a recombinant feline herpesvirus” is alive feline herpesvirus which has been generated by the recombinantmethods well known to those of skill in the art, e.g., the methods setforth in HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANTHERPESVIRUS Materials and Methods, and the virus has not had geneticmaterial essential for the replication of the feline herpesvirusdeleted.

This invention provides a recombinant feline herpesvirus comprising aforeign DNA inserted within a non-essential region of the felineherpesvirus genome, wherein the foreign DNA is inserted into a regioncorresponding to a 5.0 kb fragment of the unique long region which iswithin a SalI H fragment of the feline herpesvirus genome.

This invention provides a recombinant feline herpesvirus comprising aforeign DNA inserted within a non-essential region of the felineherpesvirus genome, wherein the foreign DNA is inserted into a SalI Mfragment of the feline herpesvirus genome.

This invention provides a recombinant feline herpesvirus comprising aforeign DNA inserted within a non-essential region of the felineherpesvirus genome, wherein the foreign DNA is inserted into SalI Nfragment of the feline herpesvirus genome.

This invention provides a recombinant feline herpesvirus furthercomprising a second foreign DNA inserted within a non-essential regionof the feline herpesvirus genome. The second foreign DNA may be insertedinto a unique short region of the feline herpesvirus genome. In oneembodiment the the second foreign DNA may be inserted into aglycoprotein E gene. In another embodiment the second foreign DNA may beinserted into a glycoprotein I gene. In another embodiment the secondforeign DNA may be inserted into a glycoprotein G gene. The secondforeign DNA may be inserted into a unique long region of the felineherpesvirus genome.

This invention provides the insertion of multiple foreign DNAs intonon-essential regions of the feline herpesvirus genome. Further, thisinvention provides multiple foreign DNAs inserted into one site orregion of the non-essential region of the feline herpesvirus genome orin different regions of the feline herpesvirus genome.

This invention provides a recombinant feline herpesvirus furthercomprising a deletion in a non-essential region of the felineherpesvirus genome. In one embodiment the deletion is in the uniqueshort region. In another embodiment the deletion is in the glycoproteinE gene. In another embodiment the deletion is in the glycoprotein Igene. In another embodiment the deletion is in a glycoprotein G gene. Inanother embodiment the deletion is in the unique long region.

As defined herein, “viral genome” or “genome” means the entire DNA whichnaturally occurrs in the feline virus. As defined herein, “foreign DNA”or “foreign gene” means any DNA or gene that is exogenous to the genome.

As defined herein “a non-essential site” or “a non-essential region” inthe feline herpesvirus genome means a region or site in the viral genomewhich is not necessary for the viral infection or replication.

In one embodiment the polypeptide is a detectable marker. For purposesof this invention, a “polypeptide which is a detectable marker” includesthe diner, trimer and tetramer form of the polypeptide. E. coliB-galactosidase is a tetramer composed of four polypeptides or monomersubunits. In one embodiment the polypeptide is E. colibeta-galactosidase.

In one embodiment the foreign DNA encodes a polypeptide. In anotherembodiment the polypeptide is antigenic. In one embodiment foreign DNAencodes Marek's Disease Virus glycoprotein A, Marek's Disease Virusglycoprotein B or Marek's Disease Virus glycoprotein D. In oneembodiment foreign DNA encodes Newcastle Disease Virus fusion protein orNewcastle Disease Virus hemagglutinin-neuraminidase.

In one embodiment foreign DNA encodes Infectious Laryngotracheitis Virusglycoprotein B, Infectious Laryngotracheitis Virus glycoprotein I orInfectious Laryngotracheitis Virus glycoprotein D. In one embodimentforeign DNA encodes Infectious Bronchitis Virus spike protein orInfectious Bronchitis Virus matrix protein. In one embodiment foreignDNA encodes Infectious Bursal Disease virus VP2, Infectious BursalDisease virus VP3, or Infectious Bursal Disease virus VP4.

In one embodiment foreign DNA is selected from a group consisting ofFeline Leukemia virus envelope gene,Feline Leukemia virus protease (gag)gene, Hepatitis B core antigen gene, Pseudorabies virus glycoprotein Cgene, Dirofilaria immitis 22 kD, Dirofilaria immitis p39 gene, FelineImmunodeficiency virus gag, Feline Immunodeficiency Virus pol gene, andFeline Immunodeficiency Virus env gene.

Such antigenic polypeptide may be derived or derivable from thefollowing: feline pathogen, canine pathogen, equine pathogen, bovinepathogen, avian pathogen, porcine pathogen, or human pathogen.

In another embodiment, the antigenic polypeptide of a human pathogen isderived from human herpesvirus, herpes simplex virus-1, herpes simplexvirus-2, human cytomegalovirus, Epstein-Barr virus, Varicell-Zostervirus, human herpesvirus-6, human herpesvirus-7, human influenza, humanimmunodeficiency virus, rabies virus, measles virus, hepatitis B virusand hepatitis C virus. Furthermore, the antigenic polypeptide of a humanpathogen may be associated with malaria or malignant tumor from thegroup consisting of Plasmodium falciparum, Bordetella pertusis, andmalignant tumor.

The antigenic polypeptide of an equine pathogen can derived from equineinfluenza virus or equine herpesvirus. Examples of such antigenicpolypeptide are equine influenza virus type A/Alaska 91 neuraminidase,equine influenza virus type A/Prague 56 neuraminidase, equine influenzavirus type A/Miami 63 neuraminidase, equine influenza virus typeA/Kentucky 81 neuraminidase, equine herpesvirus type 1 glycoprotein B,and equine herpesvirus type 1 glycoprotein D.

The antigenic polypeptide of derived from bovine respiratory syncytialvirus equine pathogen can derived from equine influenza virus is bovinerespiratory syncytial virus attachment protein (BRSV G), bovinerespiratory syncytial virus fusion protein (BRSV F), bovine respiratorysyncytial virus nucleocapsid protein (BRSV N), bovine parainfluenzavirus type 3 fusion protein, and the bovine parainfluenza virus type 3hemagglutinin neuraminidase

In one embodiment the polypeptide is derived or derivable from the groupconsisting of: avian encephalomyelitis virus, avian reovirus, avianparamyxovirus, avian influenza virus, avian adenovirus, fowl pox virus,avian coronavirus, avian rotavirus, chick anemia virus (agent),Salmonella spp. E. coli, Pasteurella spp., Bordetella spp., Eimeriaspp., Histomonas spp., Trichomonas spp., Poultry nematodes, cestodes,trematodes, poultry mites/lice, poultry protozoa.

In one embodiment foreign DNA encodes a cytokine. In another embodimentthe cytokine is chicken myelomonocytic growth factor (cMGF), chickeninterferon (cIFN) or quail interferon. For example, the cytokine may be,but is not limited to, interleukin-1-interleukin-15, interferons, quailinterferon, chicken intereferon, granulocyte-macrophage colonystimulating factors, granulocyte colony stimulating factors andinterleukin receptors, TNF, Interferon, and Stem Cell Factor.

In one embodiment foreign DNA is under control of an endogenous upstreampromoter. In another embodiment foreign DNA is under control of aheterologous upstream promoter. In another embodiment the promoter isselected from the group consisting of: Feline Herpesvirus glycoprotein Epromoter, Pseudorabies virus glycoprotein X promoter, Herpes SimplexVirus-1 alpha 4 promoter, Human Cytomegalovirus immediate earlypromoter, Marek's Disease Virus glycoprotein A promoter, Marek's DiseaseVirus glycoprotein B promoter, Marek's Disease Virus glycoprotein Dpromoter, Infectious Laryngotracheitis Virus glycoprotein B promoter,Infectious Laryngotracheitis Virus glycoprotein D promoter, and BovineHerpesvirus-1.1 VP8 promoter and chicken anemia virus promoter.

This invention provides a recombinant feline herpesvirus designatedS-FHV-020 (ATCC Accession Number VR 2573). This invention provides arecombinant feline herpesvirus of claim 1 designated S-FHV-025. Thisinvention provides a recombinant feline herpesvirus of claim 1designated S-FHV-026.

S-FHV-020 was deposited on May 9, 1997 under ATCC Accession No. VR 2573pursuant to the Budapest Treaty on the International Deposit ofMicroorganisms for the Purposes of Patent Procedure with the PatentCulture Depository of the American Type Culture Collection, 12301Parklawn Drive, Rockville, Md. 20852 U.S.A.

This invention provides a recombinant herpesvirus comprising a foreignDNA inserted into a herpesvirus genome, wherein the foreign DNA isinserted into a non-essential region and is under the control of afeline herpesvirus glycoprotein E promoter. In one embodiment theforeign DNA is capable of being expressed in a host cell into which thevirus is introduced. In one embodiment the herpesvirus is a felineherpesvirus.

This invention provides a recombinant feline herpesvirus comprising afeline herpesvirus genome, wherein the feline herpesvirus genomecontains a deletion in a region corresponding to a 3.0 kb EcoRI-SalIfragment of the SalI A fragment of the feline herpesvirus genome.

In one embodiment the deletion is in a UL25 gene within a 3.0 kbEcoRI-SalI fragment. In another embodiment the deletion is in a regionwhich correspond to a SacII—SacII fragment within the 3.0 kb EcoRI-SalIfragment. This invention provides for three such regions whichcorrespond to a SacII-SacII fragment within the 3.0 kb EcoRI-SalIfragment. For example, a region is between nucleotide sequence 1049-1361of SEQ.ID. No: 3; a region is between nucleotide sequence 1361-1928 ofSEQ.ID. No: 3; and a region is between nucleotide sequence 1049-1928 ofSEQ.ID. No: 3.

This invention provides a recombinant feline herpesvirus furthercomprising an insertion of a foreign DNA into a non-essential region ofthe feline herpesvirus genome. In another embodiment this inventionprovides a recombinant feline herpesvirus wherein the foreign DNA is afusion protein of two or more foreign DNAs.

The present invention provides a recombinant feline herpesviruscomprising a feline herpesvirus genome which contains a deletion in theunique short region of the viral genome, wherein the deletion is in theglycoprotein E gene. Said recombinant feline herpesvirus contains adeletion which attenuates the virus, rendering it suitable for use as avaccine against feline herpesvirus.

In one embodiment the feline herpesvirus contains a foreign DNA insertedinto a non-essential region of the feline herpesvirus genome. In anotherembodiment the foreign DNA is inserted into a unique short region of thefeline herpesvirus. In another embodiment the foreign DNA sequence isinserted in the deleted gE gene.

The present invention further provides a recombinant feline herpesviruscomprising the feline herpesvirus viral genome which contains a deletionor other alteration in the unique short region of the viral genome,wherein the deletion or alteration is in the glycoprotein gE gene, sothat upon replication, the recombinant virus produces no glycoproteingE.

The present invention further provides a recombinant feline herpesviruscomprising the feline herpesvirus viral genome which contains a deletionor other alteration in the unique short region of the viral genome,wherein the deletion or alteration is in the glycoprotein gI gene, sothat upon replication, the recombinant virus produces no glycoproteingI.

The present invention further provides a recombinant feline herpesviruscomprising the feline herpesvirus viral genome which contains a deletionor other alteration in the unique short region of the viral genome,wherein the deletion or alteration is in the glycoprotein gG gene and inthe glycoprotein gI gene, so that upon replication, the recombinantvirus produces no glycoprotein gG and no glycoprotein gI.

It is contemplated that a deletion in any one the above genes willattenuate the virus, rendering it suitable to be used as a vaccineagainst feline herpesvirus. A foreign DNA sequence may be insertedwithin any one of these sites in such a way that it may be expressed ina host cell which is infected which the recombinant feline herpesvirusof the present invention.

The present invention further provides a homology vector for producing arecombinant feline herpesvirus by inserting a foreign DNA into thefeline herpesvirus genome which comprises a double-stranded DNA moleculeconsisting of: a) double-stranded foreign DNA sequence encoding anantigenic polypeptide derived from a feline pathogen; b) at one end ofthe foreign DNA, double-stranded feline virus genome homologous to thegenome located at one side of a non-essential site of the feline herpesviral genome; c) at the other end of the foreign DNA sequence, doublestranded feline herpesvirus genome homologous to the genome located atthe other side of the same site.

For purposes of this invention, a “homology vector” is a plasmidconstructed to insert foreign DNA in a specific site on the genome of afeline herpesvirus.

It is contemplated by this invention that the homology vector includethe foreign DNA and antigenic polypeptides which are listed hereinabove.

For example, the double stranded foreign DNA of the homology vectorencodes an antigenic polypeptide derived from bovine respiratorysyncytial virus or bovine parainfluenza virus. The antigenic polypeptideof derived from bovine respiratory syncytial virus equine pathogen canderived from equine influenza virus is bovine respiratory syncytialvirus attachment protein (BRSV G), bovine respiratory syncytial virusfusion protein (BRSV F), bovine respiratory syncytial virus nucleocapsidprotein (BRSV N), bovine parainfluenza virus type 3 fusion protein, andthe bovine parainfluenza virus type 3 hemagglutinin neuraminidase.

In another embodiment the double stranded foreign DNA in the homologyvector encodes a cytokine capable of stimulating human immune response.For example, the cytokine may be, but is not limited to, interleukin-2,interleukin-6, interleukin-12, interferons, granulocyte-macrophagecolony stimulating factors, and interleukin receptors.

The present invention further provides a homology vector for producing arecombinant feline herpesvirus by inserting a foreign DNA into thefeline herpesvirus genome which comprises a double-stranded DNA moleculeconsisting of: a) double-stranded foreign DNA encoding an antigenicpolypeptide derived from a cytokine capable of stimulating an immuneresponse; b) at one end of the foreign DNA, double-stranded felineherpesvirus genome homologous to the genome located at one side of anon-essential site of the feline herpesvirus genome; c) at the other endof the foreign DNA, double stranded feline virus genome homologous tothe genome located at the other side of the same site.

The present invention further provides a host cell infected with therecombinant feline herpesvirus. In one embodiment the host cell is amammalian cell. Other host cells are known to those skilled in the art.

The present invention further provides a vaccine for feline herpesviruswhich comprises an effective immunizing amount of the recombinant felineherpesvirus and a suitable carrier. In one embodiment the vaccineagainst an feline pathogen comprises an effective immunizing amount ofthe recombinant feline herpesvirus and a suitable carrier. This vaccinemay contain either inactivated or live recombinant virus.

Suitable carriers for the recombinant virus are well known to thoseskilled in the art and include but are not limited to proteins, sugars,etc. One example of such a suitable carrier is a physiologicallybalanced culture medium containing one or more stabilizing agents suchas hydrolyzed proteins, lactose, etc. Preferably, the live vaccine iscreated by taking tissue culture fluids and adding stabilizing agentssuch as stabilizing, hydrolyzed proteins. Preferably, the inactivatedvaccine uses tissue culture fluids directly after inactivation of thevirus.

The present invention further provides a method of immunizing an animalagainst a human pathogen which comprises administering to the animal aneffective immunizing dose of the feline herpes vaccine. In oneembodiment the method of immunizing an animal against an feline pathogencomprises administering to the animal an effective immunizing dose ofthe feline herpes vaccine.

This invention provides a vaccine which comprises an effectiveimmunizing amount of the recombinant feline herpesvirus and a suitablecarrier. In one embodiment the carrier is a physiologically balancedculture medium containing stabilizing agents.

The present invention further provides a multivalent vaccine for felineherpesvirus and for one or more of other feline diseases which comprisesan effective immunizing amount of a recombinant virus comprising thefeline herpesvirus viral genome which contains a deletion in the uniqueshort region, wherein the deletion is in the glycoprotein E gene, and aninsertion of a foreign gene into a non-essential site of the viralgenome.

The present invention further provides a vaccine which comprises asuitable carrier and an effective immunizing amount of a recombinantfeline herpesvirus comprising the feline herpesvirus viral genome whichcontains a deletion or other alteration in the unique short region ofthe viral genome, wherein the deletion or alteration is in theglycoprotein E gene so that upon replication, the recombinant virusproduces no glycoprotein E.

For purposes of this invention, an “effective immunizing amount” of therecombinant feline herpesvirus of the present invention is within therange of 10³ to 10⁹ PFU/dose. In another embodiment the immunizingamount is 10⁵ to 10⁷ PFU/dose. In a preffered embodiment the immunizingamount is 10⁶ PFU/dose.

The method comprises administering to the animal an effective immunizingdose of the vaccine of the present invention. The vaccine may beadministered by any of the methods well known to those skilled in theart, for example, by intramuscular, subcutaneous, intraperitoneal orintravenous injection. Alternatively, the vaccine may be administeredintranasally or orally, intradermal, inovo, or ocularly.

This invention provides a method of immunizing an animal against ananimal pathogen which comprises administering to the animal an effectiveimmunizing dose of the vaccine. This invention provides a multivalentvaccine which comprises an effective immunizing amount of therecombinant feline herpesvirus.

The present invention also provides a method of immunizing an animal,wherein the animal is a feline, canine, ovine, bovine, caprine, swine orhuman. For purposes of this invention, this includes immunizing theanimal against the virus or viruses which cause the disease or diseasesfeline herpesvirus.

The present invention further provides a method of distinguishing ananimal vaccinated with a feline herpesvirus from an animal infected witha naturally-occuring feline herpesvirus which comprises analysing asample of a body fluid from the animal for the presence of felineherpesvirus gE and at least one other antigen normally expressed in ananimal infected by a naturally-occuring feline herpesvirus, determiningwhether the antigen and gE are present in the body fluid, the presenceof the antigen and the absence of gE indicative of an animal vaccinatedwith the vaccine and not infected with a naturally-occuring felineherpesvirus.

In one embodiment the presence of the antigen and of gE in the bodyfluid is determined by detecting in the body fluid antibodies specificfor the antigen and for gE.

The present invention provides a method for testing a feline todetermine whether the feline has been vaccinated with the vaccine of thepresent invention, particularly the embodiment which contains therecombinant feline herpesvirus S-FHV-020 or S-FHV-025.

This invention is further illustrated in the Experimental Detailssection which follows. This section is set forth to aid in anunderstanding of the invention but is not intended to, and should not beconstrued to, limit in any way the invention as set forth in the claimswhich follow thereafter.

EXPERIMENTAL DETAILS:

Materials and Methods

PREPARATION OF FHV VIRUS STOCK SAMPLES:S-FHV-000 was obtained from theATCC (ATCC No. 636) and S-FHV-001 was obtained from the NVSL (NVSLChallange Virus Strain SGE, Lot KS). FHV virus stock samples wereprepared by infecting Crandell Feline Kidney (CRFK) cells at amultiplicity of infection of 1.0 PFU/cell in Dulbecco's Modified EagleMedium (DMEM) containing 2 mM glutamine, 100 units/ml penicillin, 100units/ml streptomycin (these components were obtained from IrvineScientific or equivalent supplier, and hereafter are referred to ascomplete DME medium) plus 5% fetal bovine serum. After cytopathic effectwas complete, the medium and cells were harvested, aliquoted and frozenat −70° C. The titers were approximately 1×10⁷ to 1×10⁸ PFU/ml.

PREPARATION OF HERPESVIRUS DNA:A confluent monolayer of CRFK cells in a25 cm² flask or 60 mm petri dish was infected with 100 ml of virussample. After overnight incubation, or when the cells were showing 100%cytopathic effect, the cells were scraped into the medium. The cells andmedium were centrifuged at 3000 rpm for 5 minutes in a clinicalcentrifuge. The medium was decanted, and the cell pellet was gentlyresuspended in 0.5 ml solution containing 0.5% NONIDET P-40® (octylphenol ethylene oxide condensate containing an average of 9 moles ofethylene oxide per molecule) (NP-40®, purchased from Sigma Chemical Co.,St. Louis, Mo.). The sample was incubated at room temperature for 10minutes. Ten ml of a stock solution of RNase A (Sigma Chemical Co., St.Louis, Mo.) were added (stock was 10 mg/ml, boiled for 10 minutes toinactivate DNAse). The sample was centrifuged to pellet nuclei. The DNApellet was removed with a pasteur pipette or wooden stick and discarded.The supernatant fluid was decanted into a 1.5 ml Eppendorf tubecontaining 25 ml of 20% sodium dodecyl sulfate (Sigma) and 25 mlproteinase-K (10 mg/ml; Boehringer Mannheim Biochemicals, Indianapolis,Ind.). The sample was mixed and incubated at 37° C. for 30-60 minutes.An equal volume of water-saturated phenol was added and the sample wasmixed briefly. The sample was centrifuged in an Eppendorf minifuge for 5minutes at full speed. The upper aqueous phase was removed to a newEppendorf tube, and two volumes of absolute ethanol were added and thetube put at −20° C. for 30 minutes to precipitate nucleic acid. Thesample was centrifuged in an Eppendorf minifuge for 5 minutes. Thesupernatant was decanted, and the pellet was air dried and rehydrated in˜16 ml H₂O. For the preparation of larger amounts of DNA, the procedurewas scaled up to start with roller bottles or 175 cm² flasks of CRFKcells. The DNA was stored in 0.01 M tris pH 7.5, 1 mM EDTA at 4° C.

MOLECULAR BIOLOGICAL TECHNIQUES:Techniques for the manipulation ofbacteria and DNA, including such procedures as digestion withrestriction endonucleases, gel electrophoresis, extraction of DNA fromgels, ligation, phosphorylation with kinase, treatment with phosphatase,growth of bacterial cultures, transformation of bacteria with DNA, andother molecular biological methods are described in [22 and 23]. Thepolymerase chain reaction (PCR) was used to introduce restrictionendonuclease sites convenient for the manipulation of various DNAs [24].In general, amplified fragments were less than 500 base pairs in sizeand critical regions of amplified fragments were confirmed by DNAsequencing. Except as noted, these techniques were used with minorvariations.

LIGATION:DNA was joined together by the action of the enzyme T4 DNAligase (BRL). Ligation reactions contained various amounts of DNA (from0.2 to 20 mg), 20 mM Tris pH 7.5, 10 mM MgCl₂, 10 mM dithiothreitol(DTT), 200 mM ATP and 20 units T4 DNA ligase in 10-20 ml final reactionvolume. The ligation proceeded for 3-16 hours at 15° C.

DNA SEQUENCING:Sequencing was performed using the USB Sequenase Kit and³⁵S-dATP (NEN). Reactions using both the dGTP mixes and the dITP mixeswere performed to clarify areas of compression. Alternatively,compressed areas were resolved on formamide gels. Templates weredouble-stranded plasmid subclones or single stranded M13 subclones, andprimers were either made to the vector just outside the insert to besequenced, or to previously obtained sequence. Alternatively, DNAsequencing was performed on the Applied Biosystems Automated SequencerModel 388A per instructions of the manufacturer using Taq DNA polymeraseand fluorescently-labelled dideoxynucleotides. The sequence obtained wasassembled and compared using DNAStar software. Subsequent manipulationand comparison of sequences obtained was performed with Superclone andSupersee programs from Coral Software and DNAStar.

SOUTHERN BLOTTING OF DNA:The general procedure for Southern blotting wasperformed as in Maniatis et al. and Sambrook et al. [22, 23]. DNA wasblotted to nitrocellulose filters and hybridized to appropriatelylabeled DNA probes. Probes for Southern blots were prepared using eitherthe Nonradioactive DNA Labeling and Detection Kit of Boehringer Mannheimor the nick translation kit of Bethesda Research Laboratories (BRL). Inboth cases the manufacturer's recommended procedures were followed.

DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUS:The method is basedupon the calcium phosphate procedure of Graham and Van der eb [25] withthe following modifications. Virus and/or Plasmid DNA were diluted to298 ml in 0.01 M Tris pH 7.5, 1 mM EDTA. Forty ml 2M CaCl₂ was addedfollowed by an equal volume of 2× HEPES buffered saline (10 gN-2-hydroxyethyl piperazine N′-2-ethanesulfonic acid (HEPES), 16 g NaCl,0.74 g KCl, 0.25 g Na₂HPO_(4—)2H₂O, 2 g dextrose per liter H₂O andbuffered with NaOH to pH 7.4). The mixture was then incubated on ice for10 minutes, and then added dropwise to an 80% confluent monolayer ofCRFK cells growing in a 60 mm petri dish under 5 ml of medium (DME plus5% fetal bovine serum). The cells were incubated 4 hours at 37° C. in ahumidified incubator containing 5% CO₂. Media on the plates wereaspirated, and cells were treated with 20% glycerol in 1×XPBS (1.15 gNa₂HPO₄, 0.2 g KH₂PO₄, 0.8 g NaCl, 0.2 g KCl per liter H₂O) for oneminute. The cells were washed three times with 5 ml of 1×PBS and thenfed with 5 ml of medium (DME plus 5% fetal bovine serum). The cells wereincubated at 37° C. as above for 3-7 days until cytopathic effect fromthe virus was 50-100%. Virus was harvested as described above for thepreparation of virus stocks. This stock was referred to as atransfection stock and was subsequently screened for recombinant virusby the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKERGENES.

HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANTHERPESVIRUS:This method relies upon the homologous recombination betweenherpesvirus DNA and plasmid homology vector DNA which occurs in tissueculture cells co-transfected with these elements. From 0.1-1.0 mg ofplasmid DNA containing foreign DNA flanked by appropriate herpesviruscloned sequences (the homology vector) were mixed with approximately 0.3mg of intact herpesvirus DNA. The DNAs were diluted to 298 ml in 0.01 MTris pH 7.5, 1 mM EDTA and transfected into CRFK cells according to theDNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUS (see above).

DIRECT LIGATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS:Ratherthan using homology vectors and relying upon homologous recombination togenerate recombinant virus, we have also developed the technique ofdirect ligation to engineer herpesviruses. In this instance, a clonedforeign gene did not require flanking herpesvirus DNA sequences but onlyrequired that it have restriction endonuclease sites available to cutout the foreign gene fragment from the plasmid vector. A compatiblerestriction enzyme was used to cut herpesvirus DNA. A requirement of thetechnique is that the restriction enzyme used to cut the herpesvirus DNAmust cut at a limited number of sites. For FHV the restriction enzymesSfiI in S-FHV-010 is appropriate. Restriction endonuclease sitespreviously introduced into herpesviruses by other methods may also beused. The herpesvirus DNA is mixed with a 30-fold molar excess ofplasmid DNA (typically 5 mg of virus DNA to 10 mg of plasmid DNA), andthe mixture is cut with the appropriate restriction enzyme. The DNAmixture is phenol extracted and ethanol precipitated to removerestriction endonucleases, and ligated together according to theligation procedure detailed above. The ligated DNA mixture is thenresuspended in 298 ml 0.01 M Tris pH 7.5, 1 mM EDTA and transfected intoCRFK cells according to the DNA TRANSFECTION FOR GENERATING RECOMBINANTVIRUS (see above). PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROMSUBGENOMIC DNA FRAGMENTS:The ability to generate herpesviruses bycotransfection of cloned overlapping subgenomic fragments is known tothose skilled in the art [26, 27]. If deletions and/or insertions areengineered directly into the subgenomic fragments prior to thecotransfection, this procedure results in a high frequency of virusescontaining the genomic alteration, greatly reducing the amount ofscreening required to purify the recombinant virus. In the first step ofthis procedure deletions are introduced into separate viruses viahomologous recombination with enzymatic marker genes as described below.The homology vector used in this step is constructed such that theenzymatic marker gene is flanked by a restriction endonuclease site thatdoes not cut FHV in the region of the DNA to be deleted. In the secondstep a library of overlapping subgenomic fragments, capable ofregenerating wild-type virus, is constructed from randomly shearedS-FHV-001 DNA. In the third step subgenomic fragments are cloned fromeach of the individual recombinant viruses containing attenuatingdeletion/marker gene insertions, which were generated in the first step.In each case the subcloned fragment corresponds in size and location toone of the wildtype subgenomic fragments constructed in the second step.This is accomplished by screening a library of randomly shearedrecombinant virus DNA subclones with probes generated from the ends ofthe appropriate wildtype subgenomic fragment. The restrictionendonuclease sites which had been engineered to flank the marker genesin the first step are now utilized to replace the marker genes in eachsubgenomic fragment with various foreign genes (such as FeLV env, FIVenv, FIV gag, D. immitis DiPLA2). In the fourth step cotransfection ofthe appropriate overlapping wild type and deletion/insertion derivedsubgenomic fragments permits the generation of recombinant FHV virusesincorporating any desired combination of deletions and/or insertions.

SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING AN ENZYMATIC MARKERGENE:When the E.coli β-galactosidase (lacZ) marker gene was incorporatedinto a recombinant virus the plaques containing recombinants werevisualized by a simple assay. The enzymatic substrate was incorporated(300 mg/ml) into the agarose overlay during the plaque assay. For thelacZ marker gene the substrate BLUOGAL™ (halogenatedindolyl-β-D-galactoside, Life Technologies, Inc., Bethesda, Md.) wasused. Virus that expressed active marker enzyme turned blue. The blueplaques were then picked onto fresh CRFK cells, and the recombinantvirus was purified by further blue plaque isolation. For recombinantvirus construction in which the strategy requires that the enzymaticmarker gene be removed, a subsequent assay involved plaque purifyingwhite plaques from a background of parental blue plaques. In both casesviruses were typically purified with three to five sequential rounds ofplaque purification.

CONSTRUCTION OF DELETION VIRUSES:The strategy used to construct virusescontaining genomic deletions involved the use of either homologousrecombination and/or direct ligation techniques. Initially a virus wasconstructed via homologous recombination, in which the DNA to be deletedwas replaced with a marker gene such as E.coli β-galactosidase (lacZ). Asecond virus was then constructed in which the marker gene wassubsequently deleted either by homologous recombination or via directligation. The advantage of this strategy is that both viruses may bepurified by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATICMARKER GENES. The first virus is purified by picking blue plaques from awhite plaque background, the second virus is purified by picking whiteplaques from a blue plaque background.

SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT FHV USING BLACK PLAQUEASSAYS: To analyze expression of foreign antigens expressed byrecombinant FHV, monolayers of CRFK cells are infected with recombinantFHV, overlaid with nutrient agarose media and incubated for 1-2 days at37° C. Once plaques have developed, the agarose overlay was removed fromthe dish, the monolayer rinsed once with PBS, fixed with 100% methanolfor 10 minutes at room temperature and the cells air dried. Afterre-hydrating the plate with PBS, the primary antibody was diluted to theappropriate dilution with PBS plus Blotto and incubated with the cellmonolayer for 2 hours to overnight at room temperature. Unbound antibodywas removed from the cells by washing four times with PBS at roomtemperature. The appropriate secondary antibody conjugate was diluted1:500 with PBS and incubated with the cells for 2 hours at roomtemperature. Unbound secondary antibody was removed by washing the cellsthree times with PBS at room temperature. The monolayer was rinsed incolor development buffer (100 mM Tris pH 9.5/100 mM NaCl/5 mM MgCl2),and incubated 10 minutes to overnight at room temperature with freshlyprepared substrate solution (0.3 mg/ml Nitro Blue tetrazolium+0.15 mg/ml5-Bromo-4-Chloro-3-Indolyl Phosphatase in color development buffer). thereaction was stopped by replacing the substrate solution with TE (10 mMTris, pH7.5/1 mM EDTA). Plaques expressing the correct antigen stainblack.

WESTERN BLOTTING PROCEDURE: Samples of lysates and protein standardswere run on a polyacrylamide gel according to the procedure of Laemnli[39]. After gel electrophoresis the proteins were transferred andprocessed according to Sambrook et al. [23]. The primary antibody wasdiluted 1:100 with 5% non-fat dry milk in Tris-sodium chloride, andSodium Azide (TSA: 6.61 g Tris-HCl, 0.97 g Tris-base, 9.0 g NaCl and 2.0g Sodium Azide per liter H₂O). The secondary antibody was a alkalinephosphatase conjugate diluted 1:1000 with TSA.

ELISA ASSAY: Indirect Elisa assay was performed using standardtechniques as described [35].

SERUM-VIRUS NEUTRALIZATION ASSAY: Sera were tested for FHV- andFeLV-specific antibodies by a microtiter technique. Test serum wasinactivated for 30 minutes at 56° C. Duplicate 2-fold dilutions of testsera were made in 96-well microtitration plates with a 25 ml pipettor.Equal volumes of virus suspension containing approximately 300 PFU wereadded to individual wells and the serum/virus mixtures were incubated at37° C. for 1 hour. 0.05 ml of a CRFK cell suspension containingapproximately 4×10⁵ cells/ml was added to each well. The presence ofantibody was indicated by the formation of a complete monolayer in 48hours.

HOMOLOGY VECTOR HOMOLOGY VECTOR 885-30.A4:The homology vector 885-30.A4was constructed for the purpose of inserting a foreign DNA into anapproximately 2968 base pair EcoRI to SalI region of the SalI A genomicfragment of the feline herpesvirus (FHV) genome the feline herpesvirus(SEQ ID NO. 3). It incorporates an E. Coli β-galactosidase gene flankedby FHV DNA. The foreign genes were inserted into an approximately 878base pair deletion (Sac II to Sac II; Nucl. 1049 t 1928, SEQ ID NO. 3)within the approximately 2968 base pair EcoRI to SalI fragment. Adetailed description of the plasmid is given in FIG. 4. Homology vector885-30.A4 was constructed from the indicated DNA sources utilizingstandard recombinant DNA techniques [22,23]. The plasmid vector isderived from an approximately 2996 base pair EcoRI to SalI restrictionendonuclease fragment of a pSP65. Fragment 1 is an approximately 1031base pair EcoRI to SacI subfragment of the FHV SalI A fragment. Fragment2 is an approximately 423 base pair Sall to BanHI subfragment of PRVBamHI to PvuII subfragment of pJF751 [28]. Fragment 4 is anapproximately 751 base pair SacII to Sall subfragment of PRV BamHI#7[29]. Fragment 5 is an approximately 1039 base pair NdeI to SalIsubfragment of the FHV SalI A fragment.

HOMOLOGY VECTOR 885-30.F9:The homology vector 885-30.F9 was constructedfor the purpose of inserting a foreign DNA into a 2968 base pair EcoRIto SalI fragment of the SalI. A genomic fragment of the FHV genome thefeline herpesvirus (SEQ ID NO. 3). It incorporates an E. Coliβ-galactosidase gene flanked by FHV DNA. The foreign genes were insertedinto a unique NdeI site (Nucl. 1305; SEQ ID NO. 3) within the 2968 basepair EcoRI to SalI fragment. A detailed description of the plasmid isgiven in FIG. 5. Homology vector 885-30. F9 was constructed from theindicated DNA sources utilizing standard recombinant DNA techniques[22,23]. The plasmid vector is derived from an approximately 2966 basepair EcoRI to SalI restriction endonuclease fragment of a pSP65.Fragment 1 is an approximately 1342 base pair EcoRI to NdeI subfragmentof the FHV SalI A fragment. Fragment 2 is an approximately 751 base pairNdeI to SalI subfragment of PRV BamHI #7[29]. Fragment 3 is anapproximately 3006 base pair BamHI to Pvull subfragment of pJF751[28].Fragment 4 is an approximately 423 base pair SalI to BamHI subfragmentof PRV BamHI #10[29]. Fragment 5 is an approximately 1659 base pair NdeIto SalI subfragment of the FHV SalI A fragment.

HOMOLOGY VECTOR 846-88.B7: The homology vector 846-88.B7 was constructedfor the purpose of deleting a portion of the gE coding region from thefeline herpesvirus and inserting a foreign DNA. The foreign DNA is underthe control of the FHV glycoprotein E promoter. A detailed descriptionof the plasmid is given in FIG. 3. It was constructed from the indicatedDNA sources utlilizing standard recombinant DNA techniques [22, 23]. Theplasmid vector is derived from an approximately 2958 base pair Asp718Irestriction endonuclease fragment of a pSP18/pSP19 fusion such that themultiple cloning site is EcoRI/SacI/Asp718I/SacI/EcoRI. Fragment 1 is anapproximately 1415 base pair Asp718I to SmaI subfragment of the FHV SalIB fragment which contains the FHV gE promoter. Fragment 2 is anapproximately 3085 base pair fragment containing the lac Z DNA codingregion was generated via PCR and synthetic primers,5′-GCAACTGCAGCAACAATGAATTCGGATCCCG-3′ (6/96.18; SEQ ID NO. 21) and5′CGTTCTGCAGCCTCTAGCTTATTCTAGATCTTT-3′ (6/96.19; SEQ ID NO. 22). Theseprimers introduced an in-frame ATG and unique Pst I sites at both endsof the fragment. Fragment 3 is an approximately 2205 base pair SalI toAsp718I subfragment of FHV EcoRI E fragment.

EXAMPLE 1

Recombinant feline herpesvirus (FHV) containing a deletion of the entiregE gene and an insertion of a foreign DNA sequence into that site willreplicate in cats and is useful as a vaccine. The ability to isolate agE-deleted FHV confirms that the FHV gE gene (SEQ ID NOs: 1 and 2) isnon essential for replication of the recombinant FHV. Recombinant FHVexpressing foreign genes for viral, bacterial or parasite antigensprotect against disease in dogs and cats. Recombinant FHV which wasisolated contains a deletion of the gE gene within the unique short andan insertion of a foreign gene into that gE site or any non-essentialsite will replicate in cats and is useful as a vaccine.

Recombinant FHV containing a deletion of the gE gene within the uniqueshort and an insertion of a foreign gene into that site is useful as avaccine. Recombinant FHV expressing foreign genes for viral, bacterial,or parasite antigens is useful as a vaccine to protect against diseasein cats, dogs, humans, horses, cattle, swine and poultry.

The sequence of the gE gene (SEQ ID NOS: 1 and 2) includes the codingsequence for the 531 amino acid open reading frame of the gE gene andalso includes the SmaI site in the FHV SalI B fragment and the SalI sitein the FHV EcoRI E fragment which define the endpoints of the deletionof the gE gene. To one side of the FHV gE deletion, homology vectors416-80.21, 644-09.A1 and 644-09.B2 contain an approximately 1415 basepair Asp718I to SmaI subfragment of FHV SalI B containing the entirecoding sequence of the gI gene (370 amino acids). To the opposite sideof the FHV gE deletion, homology vector 416-80.21 contains anapproximately 5200 base pair SalI to PvuII subfragment of the FHV EcoRIE fragment which contains unique short and terminal repeat sequences. Tothe opposite side of the FHV gE deletion in homology vectors 644-09.A1and 644-09.B2, an approximately 2205 base pair SalI to Asp718Isubfragment of the FHV EcoRI E fragment contains unique short andterminal repeat sequences.

EXAMPLE 2

Feline Herpesvirus Unique Long insertion sites: S-FHV-025 and S-FHV-026.

Feline Herpesvirus DNA (NVSL strain, also designated as S-FHV-001) wasdigested with SalI and the resulting fragments were cloned into the SalIsite of pSP 65 (Promega Corp.) plasmid cloning vector. Plasmid386-42.1B, a DNA clone identified as the SalI A fragment (˜16 kb) of theFHV genome, was double digested with EcoRI and SalI , to createsubfragments. A 3.0 kb EcoRI/Sal I subfragment of SalI A was isolatedand subcloned, resulting in plasmid 860-85.A29. The 3.0 kb FHV EcoRI toSalI subfragment from this clone was sequenced by the method offluorescent automated sequencing (ABI).

DNA sequence homology analysis determined that regions in the 3.0 kbEcoRI to SalI FHV subfragment contain homology to HSV-1 UL24, UL25 andthe 5′ region of UL26. The order of the open reading frames is: FHVthymidine kinase (TK) gene, UL24, UL25, UL26, and FHV gB gene.

A gene cassette containing the E.coli β-galactosidase coding region withthe PRV gX promoter and the PRV gX polyadenylation regulatory signalswas inserted into 860-85.A29 to facilitate the construction of two novelFHV homology vectors. One FHV homology vector designated 885-30.A4,contains gX promoter-lacZ-gX poly A inserted into a unique NdeI site. Asecond homology vector designated 885-30.F9 contains gX promoter-lacZ-gXpoly A inserted into an 878 bp SacII deletion. The Nde I site and Sac IIdeletion interrupt a predicted open reading frame which has homology toHSV-1 UL 25.

Wild type FHV viral DNA (NVSL), combined with plasmid DNA, 885-30.A4 or885-30.F9 was introduced into CRFK cells by the calcium phosphatetransfection method. Recombinant plaques expressing β-galactosidase wereidentified from cotransfection stocks and isolated. Recombinant viruses,S-FHV-025 and S-FHV-026, are purified by several rounds of plaquepassage and isolation.

S-FHV-025

S-FHV-025 is a recombinant feline herpesvirus that has an insertion of aforeign gene into a 3.0 kb EcoRI to SalI region of the SalI A genomicfragment of the FHV genome. The foreign DNA is inserted within adeletion of 878 base pairs between SacII sites (nucleotide 1049-1928,SEQ. ID. NO: 3) within the 3.0 kb EcoRI to SalI region. The E. coli lacZ(β-galactosidase) gene is under the transcriptional control of the PRVgX promoter.

S-FHV-025 was derived from S-FHV-001 (NVSL strain). This wasaccomplished utilizing the homology vector 885-30.A4 (see Materials andMethods) and virus S-FHV-001 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT HERPESVIRUS. The transfection stock wasscreened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATICMARKER GENES. The result of multiple rounds of blue plaque purificationis the recombinant virus designated S-FHV-025.

This virus is characterized by restriction endonuclease mapping and theSOUTHERN BLOTTING DNA procedure to confirm the insertion of the E. colilacZ (β-galactosidase) gene. The virus is further characterized by BLACKPLAQUE ASSAY and WESTERN BLOT to confirm expression of the foreign DNA.S-FHV-025 is useful as a vaccine against feline rhinotracheitis and as avector to express foreign DNA from disease-causing microorganisms (seeExample 4) including but not limited to feline immunodeficiency virusand feline leukemia virus.

S-FHV-026

S-FHV-026 is a recombinant feline herpesvirus that has an insertion of aforeign gene into a 3.0 kb EcoRI to SalI region of the SalI A genomicfragment of the FHV genome. The foreign DNA is inserted into a uniqueNdeI site (nucelotide 1304-1309 SEQ. ID. NO:3) in the 3.0 kb EcoRI toSalI region. The E. Coli lacZ (β-galactosidase) gene is under thetranscriptional control of the PRV gX promoter.

S-FHV-026 was derived from S-FHV-001 (NVSL strain). This wasaccomplished utilizing the homology vector 885-30.F9 (see Materials andMethods) and virus S-FHV-001 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT HERPESVIRUS. The transfection stock wasscreened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATICMARKER GENES. The result of multiple rounds of blue plaque purificationis the recombinant virus designated S-FHV-026.

This virus is characterized by restriction endonuclease mapping and theSOUTHERN BLOTTING DNA procedure to confirm the insertion of the E. colilacZ (β-galactosidase) gene and the deletion of the 878 base pair regionbetween SacII sites. The virus is further characterized by BLACK PLAQUEASSAY and WESTERN BLOT to confirm expression of the foreign DNA.

S-FHV-026 is useful as a vaccine against feline rhinotracheitis and as avector to express foreign DNA from disease-causing microorganisms (seeExample 4) including but not limited to feline immunodeficiency virusand feline leukemia virus.

EXAMPLE 3

Recombinant feline herpesvirus (FHV) expressing a foreign DNA sequenceunder the control of the FHV glycoprotein E promoter. An approximately1415 base pair Asp718I to SmaI subfragment of the FHV SalI B fragmentcontains the FHV gE promoter. The FHV gE promoter is useful as anendogenous promoter in feline herpesvirus or as a heterologous promoterin other herpesviruses, such as pseudorabies virus, herpesvirus ofturkeys, bovine herpesvirus, equine herpesvirus, canine herpesvirus,infectious laryngotracheitis virus, human cytomegalovirus, orpoxviruses, such as fowlpox, canarypox, swinepox, raccoonpox, orvaccinia, or adenoviruses, or retroviruses.

S-FHV-020

S-FHV-020 is a recombinant feline herpesvirus that has a deletion of thegE gene and an insertion of one foreign gene at the gE deletion site.The E. coli lacZ (β-galactosidase) gene is under the transcriptionalcontrol of the FHV glycoprotein E (gE) promoter.

S-FHV-020 was derived from S-FHV-001 (NVSL strain). This wasaccomplished utilizing the homology vector 846-88.B17 (see Materials andMethods) and virus S-FHV-001 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT HERPESVIRUS. The transfection stock wasscreened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATICMARKER GENES. The result of multiple rounds of blue plaque purificationis the recombinant virus designated S-FHV-026.

S-FHV-020 is useful as a vaccine against feline rhinotracheitis and as avector to express foreign DNA from disease-causing microorganisms (seeExample 4) including but not limited to feline immunodeficiency virusand feline leukemia virus. FHV 020 is useful as a parent virus togenerate new FHV recombinants which do not contain a lac Z reportergene.

EXAMPLE 4

Recombinant feline herpesvirus expressing antigens from disease causingmicroorganisms. In addition to recombinant feline herpesvirus expressingantigens from feline leukemia virus, feline immunodeficiency virus andDirofilaria immitis (heartworm). Additional antigens from diseasecausing microorganisms in cats include, but art not limited toDirofilaria immitis p39 and 22 kD antigens, feline infectiousperitonitis virus, calicivirus, rabies, virus, feline parvovirus(panleukopenia virus), feline coronavirus and feline Chlamydia,Toxoplasma gondii. Disease causing microorganisms in dogs include, butare not limited to canine distemper, canine adenovirus type 1(hepatitis), adenovirus type 2 (respiratory disease), parainfluenza,leptospira canicola, icterohemorragia, parvovirus, coronavirus, borreliaburgdorferi, canine herpesvirus, bordetella bronchiseptica and rabiesvirus.

Recombinant feline herpesviruses are useful for expressing antigens fromdisease causing microorganisms from animals in addition to dogs andcats. Recombinant feline herpesvirus is useful as a vaccine in animalsincluding but not limited to humans, horses, cattle, swine and poultry.

Recombinant feline herpesvirus is useful as a vaccine against equinediseases when foreign antigens from the following diseases or diseaseorganisms are expressed in the feline herpesvirus vector: equineinfluenza, equine herpesvirus-1 and equine herpesvirus-4. Recombinantfeline herpesvirus is useful as a vaccine against bovine diseases whenforeign antigens from the following diseases, or disease organisms areexpressed in the feline herpesvirus vector: bovine viral diarrhea virus,bovine respiratory syncytial virus, bovine parainfluenza virus.Recombinant feline herpesvirus is useful as a vaccine against swinediseases when foreign antigens from the following diseases or diseaseorganisms are expressed in the feline herpesvirus vector: pseudorabiesvirus, porcine reproductive and respiratory syndrome (PRRS/SIRS), hogcholera virus, swine influenza virus, swine parvovirus, swine rotavirus.Recombinant feline herpesvirus is useful as a vaccine against poultrydiseases when foreign antigens from the following diseases or diseaseorganisms are expressed in the feline herpesvirus vector: infectiousbronchitis virus, Newcastle disease virus, infectious bursal diseasevirus, Marek's disease virus, infectious laryngotracheitis virus.

Recombinant feline herpesvirus is useful as a vaccine against humandiseases. For example, human influenza is a rapidly evolving virus whoseneutralizing viral epitopes are rapidly changing. A useful recombinantfeline herpesvirus vaccine is one in which the influenza neutralizingeptiopes are quickly changed to protect against new strains ofinfluenza. Human influenza HA and NA genes are cloned into therecombinant feline herpesvirus. Recombinant feline herpesvirus is usefulas a vaccine against other human diseases when foreign antigens from thefollowing diseases or disease organisms are expressed in the felineherpesvirus vector: hepatitis B virus surface and core antigens,hepatitis C virus, herpes simplex virus, human herpesviruses, herpessimplex virus-1, herpes simplex virus-2, human herpesvirus-6, humanherpesvirus-7, human cytomegalovirus, Epstein-Barr virus,Varicella-Zoster virus, human immunodeficiency virus, human influenza,measles virus, hantaan virus, pneumonia virus, rhinovirus, poliovirus,human respiratory syncytial virus, retrovirus, human T-cell leukemiavirus, rabies virus, mumps virus, malaria (Plasmodium falciparum),Bordetella pertussis, Diptheria, Rickettsia prowazekii, Borreliabergdorferi, Tetanus toxoid, malignant tumor antigens.

Recombinant feline herpesviruses coexpressing a species-specificcytokine and an antigen from a disease causing microorganism are usefulto stimulate an increased cellmediated and humoral immune response inthe animal and increases the efficacy of the recombinant felineherpesvirus as a vaccine. Cytokines which are expressed in felineherpesvirus include but are not limited to interleukin-1 throughInterleukin-18, interferon, B7-1, B7-2 and granulocyte-macrophage colonystimulating factor.

REFERENCES

1. R. W. Price and A. Kahn, Infection and Immunity, 34, 571-580 (1981).

2. R. B. Tenser, et al., Journal of Clinical Microbiology 17, 122-127(1983).

3. B. Roizman, et al., Cold Spring Harbor Conference on New Approachesto Viral Vaccines (September 1983).

4. K. Fukuchi et al., Proc. Natl. Acad. Sci. U.S.A. 82, 751-754 (1985).

5. J. M. Koomey et al., Journal of Virology 50, 662-665 (1984).

6. B. Lomniczi et al., Journal of Virology 49, 970-979 (1984).

7. R. A. Crandell and F. D. Maurer, Proc. Soc. Exp. Biol. Med. 97,487-490 (1958).

8. R. M. Gaskell and R. C. Povey, Res. Vet. Sci. 27 167-174 (1978).

9. R. C. Povey, Microbiol. Infect. Dis. 2 373-387 (1979).

10. K. Tham and M. Studdert, Veterinary Record 120 321-326.

11. F. Fenner,. P. A. Bachmann, E. P. J. Gibbs, F. A. Murphy, M. J.Studdert and D. O. White, Veterinary Virology, Academic Press, Inc.,(1987).

12. A. Grail, D. A. Harbour, W. Chia, Arch. Virology 116, 209-220(1991).

13. P. A. Rota, R. K. Maes and W. T. Ruyechan, Virology 154, 168-179(1986).

14. J. H. Nunberg, et al., J. Virology 63, 3240-3249 (1989).

15. Federal Register, Vol. 55, No. 90, pp. 19245-19253

16. R. W. Honess, Journal of General Virology 65, 2077-2107 (1984).

17. M. L. Cook & J. G. Stevens, Journal of General Virology 31, 75-80(1976).

18. S. Joshi, et al., Journal of Virology 65, 5524-5530 (1991).

19. M. Wachsman, et al., Journal of General Virology 70, 2513-2520(1989).

20. R. A. Bhat, et al., Nucleic Acids Research 17, 1159-1176 (1989)

21. J. H. Elder and J. I. Mullins, Journal of Virology 46 871-880(1983).

22. T. Maniatis, et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor, N.Y. (1982).

23. J. Sambrook, et al., Molecular Cloning: A Laboratory Manual SecondEdition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989).

24. M. A. Innis et al. PCR Protocols: A Guide to Methods andApplications, Academic Press, Inc., San Diego (1990).

25. Graham and Van der eb (1973), Virology 52, 556-557 (1973).

26. M. Zijil, et al., Journal of Virology 62, 2191-2195 (1988).

27. M. A. Wild, et al., 15th International Herpesvirus Workshop,Abstract No. 122, Washington, D.C. (1990).

28. F. A. Ferrari, et al., Journal of Bacteriology 161, 556-562 (1985).

29. B. Lomniczi et al., Journal of Virology 49 970-979 (1984).

30. D. J. McGeoch et al., Journal of Molecular Biology 181 1-13 (1985).

31. D. R. Thomsen et al., Gene 16 207-217 (1981).

32. T. R. Phillips, et al., J. Virology 64 4605-4613 (1990).

33. J. P. Ryan, et al., J. Virology 61 2962-2972 (1987).

34. J. A. Culpepper, et al., Mol. Biochem. Parasitol. 54 51-62 (1992).

35. J. E. Coligan, ed., Current Protocols in Immunology John Wiley &Sons, Inc. (1994)

36. G. C. Cole, et al., J. Virology 64 4930-4938 (1990).

37. R. L. Burke, Seminars in Virology 4 187-197 (1993).

38. T. C. Mettenleiter, Comparative Immunology, Microbiology andInfectious Diseases 14 151-163 (1991).

39. U. K. Laemnli, Nature 227, 680-685 (1970)

40. Willemse, et al., European application EP 576 092 A1.

41. Nunberg, et al., U.S. Pat. No. 5,324,664.

42. Post, et al., PCT International application WO 90/01547.

43. Sondermeijer, et al., PCT International application WO 94/03621

44. Maes, et al., PCT International application WO 95/00172

45. Willemse, et al., Journal of General Virology, 75, 3107-3116 (1994).

23 1 2020 DNA feline herpesvirus 1 1 ggatccatca tcaatagtgc aatacgcaagcatataatgg tctgtgctgg gcggcggatc 60 tatataccaa acaacgatgg gcgaccatcaacggaaatga cacggtttac tcgccagact 120 aaaccatcga attcgagttc caagtccctactcgatgtcc ccagatcttc gaattccacc 180 ccaaccgatg gcgtctctag aagtcagttaaccgtaatta acgaagaaac ctaatatatt 240 tataaacaaa taaaatactt ttaaaaatggatatctggtc atgtgtaatg ttgacgcata 300 gtgggtggtg acctaagatt atattaaaatgtagaaggtt ttatgcccag ttcacagtat 360 ctactgtgac ctaccccggg gtggtaataacaatactatc gaatagccaa caatgggact 420 gcttgttacc atcctcgtga tattattgattgttacttca tcaagttcta ctattcatca 480 agtaacgatg acagaaggtg ccgcacttttagtcgatggg gatgggatcg acccaccttt 540 aaacaaaact tcacattttt tgcgaggttggacatttcta gagactccga aaggatgtac 600 aggagaggtg agtgttctaa aagtatgtatagatcgtggg gtatgtccgg atgatatcgt 660 tataaataag agatgtggtc acaaaatgcttgaaacccca ctagcgttgg cggaatttgg 720 aatttctaat agttctctca tcagaaccaaagacgtatat ttcgtgaata agaccgtgtt 780 tccaattctc acacccgaaa aaagtggccttggtattcag ggggccacta cgaatatatc 840 cgggatatat accctgcatg agcacggtgataatggatgg agtcatcaat ctacattttt 900 tgtgaccgta aaggcaaaac atcccggaccatcgttaacc ccagcaccgg ttcacttaat 960 aacaccacat cgccatgggg cacatttccacgtaagaaac tatcattcgc atgtctacat 1020 tccgggagat aagttcttat tagaaatgcacctcaaatca gatatctatg atccagaatt 1080 ttcagcaaca atagactggt attttatggagactgatata aaatgcccag tttttagaat 1140 ttatgaaact tgtatatttc acccccatgccgcatcctgt ctacatccgg aagatccctc 1200 atgcagtttt acatcaccac ttcgagcggtatctttaatt aatagatttt atccaaaatg 1260 cgatcacaga tatgccgatt ggacatccagatgtatcaac actccaagta taaatcatat 1320 gccatatatc gaacagccgg ccaataacgtggatctaaag tttatcaatg tacccaccaa 1380 cgcttctggg ttgtacgtat tcatacttcgttataatgga catccggaag aatggaccta 1440 tacactcata tcaacaggag ctaaatttttgaatgtgatt agggatctga cacgcccacg 1500 tcttggtagt catcaaatag agaccgatattagcacatct tcgcagtcgc ctaccacgga 1560 gacaccacga aacatacata taacgtgggcgagacgttat ctaaaggtta tcataggaat 1620 aatttgcgta gctggtatcc ttttgattgtaatctctatc acatgttata ttcgatttcg 1680 tcatatgcga tataaaccat atgaagtgatcaacccattc cctgcggtat ataccagcat 1740 tcctagtaac gatcccgacg aactctactttgaacgtatc gcatcgaacg acgaagaatc 1800 ggcagatgat tcttttgatg aatcagatgaggaggagcca ttgaataatc atcatatttc 1860 aacaacccaa catactgata ttaatccagaaaaatccgga tctgggtaca gtgtatggtt 1920 tcgtgataca gaagatacat cacctcagcccctacacgct cctccagatt acagtcgcgt 1980 agttaaaaga ttaaagtcta ttttaaaatgacccgtcgac 2020 2 532 PRT feline herpesvirus 1 2 Met Gly Leu Leu Val ThrIle Leu Val Ile Leu Leu Ile Val Thr Ser 1 5 10 15 Ser Ser Ser Thr IleHis Gln Val Thr Met Thr Glu Gly Ala Ala Leu 20 25 30 Leu Val Asp Gly AspGly Ile Asp Pro Pro Leu Asn Lys Thr Ser His 35 40 45 Phe Leu Arg Gly TrpThr Phe Leu Glu Thr Pro Lys Gly Cys Thr Gly 50 55 60 Glu Val Ser Val LeuLys Val Cys Ile Asp Arg Gly Val Cys Pro Asp 65 70 75 80 Asp Ile Val IleAsn Lys Arg Cys Gly His Lys Met Leu Glu Thr Pro 85 90 95 Leu Ala Leu AlaGlu Phe Gly Ile Ser Asn Ser Ser Leu Ile Arg Thr 100 105 110 Lys Asp ValTyr Phe Val Asn Lys Thr Val Phe Pro Ile Leu Thr Pro 115 120 125 Glu LysSer Gly Leu Gly Ile Gln Gly Ala Thr Thr Asn Ile Ser Gly 130 135 140 IleTyr Thr Leu His Glu His Gly Asp Asn Gly Trp Ser His Gln Ser 145 150 155160 Thr Phe Phe Val Thr Val Lys Ala Lys His Pro Gly Pro Ser Leu Thr 165170 175 Pro Ala Pro Val His Leu Ile Thr Pro His Arg His Gly Ala His Phe180 185 190 His Val Arg Asn Tyr His Ser His Val Tyr Ile Pro Gly Asp LysPhe 195 200 205 Leu Leu Glu Met His Leu Lys Ser Asp Ile Tyr Asp Pro GluPhe Ser 210 215 220 Ala Thr Ile Asp Trp Tyr Phe Met Glu Thr Asp Ile LysCys Pro Val 225 230 235 240 Phe Arg Ile Tyr Glu Thr Cys Ile Phe His ProHis Ala Ala Ser Cys 245 250 255 Leu His Pro Glu Asp Pro Ser Cys Ser PheThr Ser Pro Leu Arg Ala 260 265 270 Val Ser Leu Ile Asn Arg Phe Tyr ProLys Cys Asp His Arg Tyr Ala 275 280 285 Asp Trp Thr Ser Arg Cys Ile AsnThr Pro Ser Ile Asn His Met Pro 290 295 300 Tyr Ile Glu Gln Pro Ala AsnAsn Val Asp Leu Lys Phe Ile Asn Val 305 310 315 320 Pro Thr Asn Ala SerGly Leu Tyr Val Phe Ile Leu Arg Tyr Asn Gly 325 330 335 His Pro Glu GluTrp Thr Tyr Thr Leu Ile Ser Thr Gly Ala Lys Phe 340 345 350 Leu Asn ValIle Arg Asp Leu Thr Arg Pro Arg Leu Gly Ser His Gln 355 360 365 Ile GluThr Asp Ile Ser Thr Ser Ser Gln Ser Pro Thr Thr Glu Thr 370 375 380 ProArg Asn Ile His Ile Thr Trp Ala Arg Arg Tyr Leu Lys Val Ile 385 390 395400 Ile Gly Ile Ile Cys Val Ala Gly Ile Leu Leu Ile Val Ile Ser Ile 405410 415 Thr Cys Tyr Ile Arg Phe Arg His Met Arg Tyr Lys Pro Tyr Glu Val420 425 430 Ile Asn Pro Phe Pro Ala Val Tyr Thr Ser Ile Pro Ser Asn AspPro 435 440 445 Asp Glu Leu Tyr Phe Glu Arg Ile Ala Ser Asn Asp Glu GluSer Ala 450 455 460 Asp Asp Ser Phe Asp Glu Ser Asp Glu Glu Glu Pro LeuAsn Asn His 465 470 475 480 His Ile Ser Thr Thr Gln His Thr Asp Ile AsnPro Glu Lys Ser Gly 485 490 495 Ser Gly Tyr Ser Val Trp Phe Arg Asp ThrGlu Asp Thr Ser Pro Gln 500 505 510 Pro Leu His Ala Pro Pro Asp Tyr SerArg Val Val Lys Arg Leu Lys 515 520 525 Ser Ile Leu Lys 530 3 2968 DNAfeline herpesvirus 1 3 gaattcgatg ccacaatcgc ttttatgctg tcctcaagcgcgatatatcc tcgacctctg 60 cgagtggagt atataccgac cgactcgagt gtttattgggcggctctgta tccgcagagt 120 ctcttaaaaa ggctaagggc gtgtacctag catgtgaggttaacttgggt cgtcgccgac 180 ctgattgtgt atgtactata caatttgagg gagaagggggtggtatatgt tttctaattg 240 agctgaaaac gtgtcgtttt tcaaaaaata tggatacaactagtaaagac attcaacgtc 300 gtgagggttt gaaacaatta acagattctg tgggtttaataaccaagatt ctaccaccag 360 ggggggagaa gctcactcta atacccatat tggcatttatcgcgcagcgc ggtctgagaa 420 ttttgggagt tactaattta cccccgcaga tgcttacgaataacatttct gttctggctg 480 ctaatattac caagctcgcc gaatacaatc caatcgagagtggtggtgta atccgctcca 540 agaaaaaatc aaaataccca aggtctgggt ctggggtctataaacatacg aaccacgtac 600 cgatgtcatc gtgtgtatct gtgaaggata aaaaaaacactttaactcca ataggaagcg 660 gcgaaagtaa ccctttaaaa tgggtagcat ctctctttcccgatcactcc gctacgcaac 720 cacgtgagta ttaatatgcc ctatatgtga tgggtaatgagtctatatag atcatatatc 780 aaaaccttat ttaggatcaa agagattaat cactggtatcatttgttgga gaggagaagt 840 tgtcgaagac gggtcatcat gactggacga gtcgaatacgtattcgactc gatgcgaata 900 actaacatcg gggatgatct gattttatcg gacactagaaattttattac tcccacattt 960 ccagtggatt attggcgtga gccaaccttt tatttaaatgaaaaaactac tcctgaaagt 1020 ctagatgtcc gccgaaaggc tgctgccgcg gccctggataatttaaccca tcaaaaacta 1080 ttgggcgaaa cggatataga ggatcgtcta tatcccttggagcaacaggt actaaatgtg 1140 gctaacgcat tggcctctct agaggaggta gcacgggaagcggaaacggc ggacgctgag 1200 atggataagg atactagacc tctccagtct aatggtggaagcagatcgga tgagacacct 1260 ggagggcttg aggttcagat taccaaaaat gacactccattggcatatga aacaaaccta 1320 gccatagatt ttttaacgat ggtatattta gggcgtgccgcgggatcaaa tggtatatca 1380 tttggttcat ggtatagagc gttacaagat cgtcttatcacagatcgtcc gttaaccacc 1440 agaagtatag attatcgaga tgggcgaatg tctaaaacttccatgacagc aacaataatg 1500 tctctccaat cctgtgcccg actctatatc gggaatagagcctactcggc ttttgaatgt 1560 gcagttttat gtcttcatct tttacatcgg gaactcgataagggaatcat gacgcaccca 1620 ccgactacat tttccgatct aatagagcac ctaccgacatcattggatat tattgctaac 1680 accctaggta ctatgccgtc tggtagagta atttatatatataatataga taaactacct 1740 agacatcaat ttcaggcgcc taatggtgga cggtatgaacatcatgccct tgaagaccat 1800 agcgtattaa atctacttct ccaatttaag gttttacctccgattcctgg acatattaaa 1860 ggtggtcccc cggctatagc catagatata gaccagactgctttcgtaga cccggttaat 1920 agagccgcgg cagctttcct agggagggca cataatctatttctcacgga ggatcagacg 1980 cttctcagag ctactataaa cactataacc tcactattactactccgacg cttactatgg 2040 aatggaaaca tctatacgga taaattacgt aataatttccagcttggaac actcataccc 2100 cagacagcct ctatacagat gctgggtaca ttgactcgcggggcaaccgg gggggatttg 2160 ggagcaccac tcaccataaa aagcgagagt cacaatctagagtttttatg ttctagatat 2220 atattaccga tctatacatc tatgccagat gtcgagatcacgcaattatt tccgggtctt 2280 acagcgctat gtttagatgc gcaagccctc atagcacaaacccgcaccgc gaggcgcgtg 2340 gtgcaagtga aaactggacg tttgcaggac aacttaattcggttggttgg tctcgaactc 2400 gaaaatagac gccggacagg aacagtaccc ataggtgaggtgatcacggc acacgacgct 2460 atatctcttc aaactgaaca tggcttgggt cttctcatgcaacaaccacg acttagagcc 2520 tctcttgagg aaaatcatcg cttatggcaa tttaacattggcagtgatta cgatctaata 2580 tactttctgt gtttggggta tatacctcaa ttcacagcatctatataact gaaatgtttt 2640 gctaagtaga tcatatacga aataaacatt acatattaaatataacacac ctggtgcgtg 2700 tgggtctttt tattttcaac cggctctcca gtaggaagcaccagtttctt cacatgcaat 2760 ggatacacac gatattatcg aggataccac atccgatctccatatctacg tggctggtta 2820 tctagccctc tatgatatgg gagatggcgg ggaattgactttaactcgtg atgtggtgcg 2880 tgcagcattg cccccagcat caccactaca gattaatatagatcataatc gtaaatgtgt 2940 catcggttcc gtactatcta ttgtcgac 2968 4 240 PRTfeline herpesvirus 1 4 Ile Arg Cys His Asn Arg Phe Tyr Ala Val Leu LysArg Asp Ile Ser 1 5 10 15 Ser Thr Ser Ala Ser Gly Val Tyr Thr Asp ArgLeu Glu Cys Leu Leu 20 25 30 Gly Gly Ser Val Ser Ala Glu Ser Leu Lys LysAla Lys Gly Val Tyr 35 40 45 Leu Ala Asn Leu Gly Arg Arg Arg Pro Asp CysVal Cys Thr Ile Gln 50 55 60 Phe Glu Gly Glu Gly Gly Gly Ile Cys Phe LeuIle Glu Leu Lys Thr 65 70 75 80 Cys Arg Phe Ser Lys Asn Met Asp Thr ThrSer Lys Asp Ile Gln Arg 85 90 95 Arg Glu Gly Leu Lys Gln Leu Thr Asp SerVal Gly Leu Ile Thr Lys 100 105 110 Ile Leu Pro Pro Gly Gly Glu Lys LeuThr Leu Ile Pro Ile Leu Ala 115 120 125 Phe Ile Ala Gln Arg Gly Leu ArgIle Leu Gly Val Thr Asn Leu Pro 130 135 140 Pro Gln Met Leu Thr Asn AsnIle Ser Val Leu Ala Ala Asn Ile Thr 145 150 155 160 Lys Leu Ala Glu TyrAsn Pro Ile Glu Ser Gly Gly Val Ile Arg Ser 165 170 175 Lys Lys Lys SerLys Tyr Pro Arg Ser Gly Ser Gly Val Tyr Lys His 180 185 190 Thr Asn HisVal Pro Met Ser Ser Cys Val Ser Val Lys Asp Lys Lys 195 200 205 Asn ThrLeu Thr Pro Ile Gly Ser Gly Glu Ser Asn Pro Leu Lys Trp 210 215 220 ValAla Ser Leu Phe Pro Asp His Ser Ala Thr Gln Pro Arg Glu Tyr 225 230 235240 5 589 PRT feline herpesvirus 1 5 Met Thr Gly Arg Val Glu Tyr Val PheAsp Ser Met Arg Ile Thr Asn 1 5 10 15 Ile Gly Asp Asp Leu Ile Leu SerAsp Thr Arg Asn Phe Ile Thr Pro 20 25 30 Thr Phe Pro Val Asp Tyr Trp ArgGlu Pro Thr Phe Tyr Leu Asn Glu 35 40 45 Lys Thr Thr Pro Glu Ser Leu AspVal Arg Arg Lys Ala Ala Ala Ala 50 55 60 Ala Leu Asp Asn Leu Thr His GlnLys Leu Leu Gly Glu Thr Asp Ile 65 70 75 80 Glu Asp Arg Leu Tyr Pro LeuGlu Gln Gln Val Leu Asn Val Ala Asn 85 90 95 Ala Leu Ala Ser Leu Glu GluVal Ala Arg Glu Ala Glu Thr Ala Asp 100 105 110 Ala Glu Met Asp Lys AspThr Arg Pro Leu Gln Ser Asn Gly Gly Ser 115 120 125 Arg Ser Asp Glu ThrPro Gly Gly Leu Glu Val Gln Ile Thr Lys Asn 130 135 140 Asp Thr Pro LeuAla Tyr Glu Thr Asn Leu Ala Ile Asp Phe Leu Thr 145 150 155 160 Met ValTyr Leu Gly Arg Ala Ala Gly Ser Asn Gly Ile Ser Phe Gly 165 170 175 SerTrp Tyr Arg Ala Leu Gln Asp Arg Leu Ile Thr Asp Arg Pro Leu 180 185 190Thr Thr Arg Ser Ile Asp Tyr Arg Asp Gly Arg Met Ser Lys Thr Ser 195 200205 Met Thr Ala Thr Ile Met Ser Leu Gln Ser Cys Ala Arg Leu Tyr Ile 210215 220 Gly Asn Arg Ala Tyr Ser Ala Phe Glu Cys Ala Val Leu Cys Leu His225 230 235 240 Leu Leu His Arg Glu Leu Asp Lys Gly Ile Met Thr His ProPro Thr 245 250 255 Thr Phe Ser Asp Leu Ile Glu His Leu Pro Thr Ser LeuAsp Ile Ile 260 265 270 Ala Asn Thr Leu Gly Thr Met Pro Ser Gly Arg ValIle Tyr Ile Tyr 275 280 285 Asn Ile Asp Lys Leu Pro Arg His Gln Phe GlnAla Pro Asn Gly Gly 290 295 300 Arg Tyr Glu His His Ala Leu Glu Asp HisSer Val Leu Asn Leu Leu 305 310 315 320 Leu Gln Phe Lys Val Leu Pro ProIle Pro Gly His Ile Lys Gly Gly 325 330 335 Pro Pro Ala Ile Ala Ile AspIle Asp Gln Thr Ala Phe Val Asp Pro 340 345 350 Val Asn Arg Ala Ala AlaAla Phe Leu Gly Arg Ala His Asn Leu Phe 355 360 365 Leu Thr Glu Asp GlnThr Leu Leu Arg Ala Thr Ile Asn Thr Ile Thr 370 375 380 Ser Leu Leu LeuLeu Arg Arg Leu Leu Trp Asn Gly Asn Ile Tyr Thr 385 390 395 400 Asp LysLeu Arg Asn Asn Phe Gln Leu Gly Thr Leu Ile Pro Gln Thr 405 410 415 AlaSer Ile Gln Met Leu Gly Thr Leu Thr Arg Gly Ala Thr Gly Gly 420 425 430Asp Leu Gly Ala Pro Leu Thr Ile Lys Ser Glu Ser His Asn Leu Glu 435 440445 Phe Leu Cys Ser Arg Tyr Ile Leu Pro Ile Tyr Thr Ser Met Pro Asp 450455 460 Val Glu Ile Thr Gln Leu Phe Pro Gly Leu Thr Ala Leu Cys Leu Asp465 470 475 480 Ala Gln Ala Leu Ile Ala Gln Thr Arg Thr Ala Arg Arg ValVal Gln 485 490 495 Val Lys Thr Gly Arg Leu Gln Asp Asn Leu Ile Arg LeuVal Gly Leu 500 505 510 Glu Leu Glu Asn Arg Arg Arg Thr Gly Thr Val ProIle Gly Glu Val 515 520 525 Ile Thr Ala His Asp Ala Ile Ser Leu Gln ThrGlu His Gly Leu Gly 530 535 540 Leu Leu Met Gln Gln Pro Arg Leu Arg AlaSer Leu Glu Glu Asn His 545 550 555 560 Arg Leu Trp Gln Phe Asn Ile GlySer Asp Tyr Asp Leu Ile Tyr Phe 565 570 575 Leu Cys Leu Gly Tyr Ile ProGln Phe Thr Ala Ser Ile 580 585 6 69 PRT feline herpesvirus 1 6 Met AspThr His Asp Ile Ile Glu Asp Thr Thr Ser Asp Leu His Ile 1 5 10 15 TyrVal Ala Gly Tyr Leu Ala Leu Tyr Asp Met Gly Asp Gly Gly Glu 20 25 30 LeuThr Leu Thr Arg Asp Val Val Arg Ala Ala Leu Pro Pro Ala Ser 35 40 45 ProLeu Gln Ile Asn Ile Asp His Asn Arg Lys Cys Val Ile Gly Ser 50 55 60 ValLeu Ser Ile Val 65 7 1730 DNA feline herpesvirus 1 7 gtcgacgaccggccatcgcg aagacaacgc ttctcgggcg atgaatgtga ataccaatgt 60 ccactaatcccgggtctttt atagggtgtc cggcattcga catgatgctg aatcgtttcc 120 ggggaaaacccccaaatttc cgagacgttt tagtgtggca tgcgtcgaag ttcgctgctt 180 accatgcgtagacacatccg gtaggcgtaa caaatcatag ccacacccac ggcaatcaag 240 gacatgtaaaataaaacagc ggatgcagtg ggggtgtttg atattcacac cccttgccga 300 acacgaagcgctccaaaatt gctttagccg ctccatactc tcaaaatctt ccgcgtttgc 360 ggggcatctgaatcgctgta gtagaagaat ggcgaccaat aggataacca gcactgatgt 420 tacggataaacgatccatct ccgttttggt agaaaagact tcagaggcgt ggagtttatt 480 ggtcagtgagaatgctctgt tggtgacaaa taatcaacca atcgagcttg ggttgatgag 540 tagagagccggataatgcgg acatctttta taccagaccg gtagatattg ggcttagatt 600 agccacgcctgatggatatg caattttaat tacgcaaacg tgctcctccc aagatcccag 660 tcaagcagtgtcaatcatca atggggttat cgattctggg tatcgcggaa ttctcaaagc 720 gctaatctaccacagaccgt gtatcgagac tattaaggag tatggtctaa agttacaact 780 acctctactcaagcttagta aagctacgat tacattagca ccatgtcctg ctacgattag 840 gcataaacaaggggtcccaa tgggtgctcg actgtgtgat ttctatgaaa tttttaaaca 900 aaaacgggacgaagatgctg gttatgatat atccgcacct gaaacgttcc agatttatcc 960 gggatttaatcactatgtag agatcccagt agttcacctt ccgggtgaca acccccccat 1020 agcatgtatctttgggaggt cttctcttaa cgtgagtggt atcgtagttc tccctactgt 1080 atggaaacctgagacaaaat gtgggttctt cataaaaaat atgcgtcgcg atccagtaat 1140 cattagagctgggcaaagga ttgcccagtt attacttctg gaagaattac ctatggaatg 1200 gctacctacggaaacgaata atcacgatcc atttccagaa accccagaac ctgcacctgg 1260 aacaatcatggctcacgcgg atttatggac ctttactgaa aacttcatcg tgatgcccca 1320 tcgagccttcggggggataa aggatttggg tcaaccgggg tataataaaa taaacgatat 1380 gataaataacataacaacga aatctgtttt tattccatag tcaattctgt ttgtcgatgt 1440 gatttgcgcttggatttttt agtatgtctt atagaatctc cattatcaaa gatatggtcg 1500 ttaaccagaaccggttcacg ggctacgacg tttggagtta tgtccatagt agtaacagat 1560 gtgacattcgggtgatttga tattttgcac tcctccgatg cggatggttg attagtaact 1620 agatgtctgttccccgagct tgcagcaccg ataccaaaag tcttctctaa tacagctaca 1680 tcggccatcacaatgttatt ttcggaactc atgcgtatgg cttggtcgac 1730 8 384 PRT felineherpesvirus 1 8 Met Gln Trp Gly Cys Leu Ile Phe Thr Pro Leu Ala Glu HisGlu Ala 1 5 10 15 Leu Gln Asn Cys Phe Ser Arg Ser Ile Leu Ser Lys SerSer Ala Phe 20 25 30 Ala Gly His Leu Asn Arg Cys Ser Arg Arg Met Ala ThrAsn Arg Ile 35 40 45 Thr Ser Thr Asp Val Thr Asp Lys Arg Ser Ile Ser ValLeu Val Glu 50 55 60 Lys Thr Ser Glu Ala Trp Ser Leu Leu Val Ser Glu AsnAla Leu Leu 65 70 75 80 Val Thr Asn Asn Gln Pro Ile Glu Leu Gly Leu MetSer Arg Glu Pro 85 90 95 Asp Asn Ala Asp Ile Phe Tyr Thr Arg Pro Val AspIle Gly Leu Arg 100 105 110 Leu Ala Thr Pro Asp Gly Tyr Ala Ile Leu IleThr Gln Thr Cys Ser 115 120 125 Ser Gln Asp Pro Ser Gln Ala Val Ser IleIle Asn Gly Val Ile Asp 130 135 140 Ser Gly Tyr Arg Gly Ile Leu Lys AlaLeu Ile Tyr His Arg Pro Cys 145 150 155 160 Ile Glu Thr Ile Lys Glu TyrGly Leu Lys Leu Gln Leu Pro Leu Leu 165 170 175 Lys Leu Ser Lys Ala ThrIle Thr Leu Ala Pro Cys Pro Ala Thr Ile 180 185 190 Arg His Lys Gln GlyVal Pro Met Gly Ala Arg Leu Cys Asp Phe Tyr 195 200 205 Glu Ile Phe LysGln Lys Arg Asp Glu Asp Ala Gly Tyr Asp Ile Ser 210 215 220 Ala Pro GluThr Phe Gln Ile Tyr Pro Gly Phe Asn His Tyr Val Glu 225 230 235 240 IlePro Val Val His Leu Pro Gly Asp Asn Pro Pro Ile Ala Cys Ile 245 250 255Phe Gly Arg Ser Ser Leu Asn Val Ser Gly Ile Val Val Leu Pro Thr 260 265270 Val Trp Lys Pro Glu Thr Lys Cys Gly Phe Phe Ile Lys Asn Met Arg 275280 285 Arg Asp Pro Val Ile Ile Arg Ala Gly Gln Arg Ile Ala Gln Leu Leu290 295 300 Leu Leu Glu Glu Leu Pro Met Glu Trp Leu Pro Thr Glu Thr AsnAsn 305 310 315 320 His Asp Pro Phe Pro Glu Thr Pro Glu Pro Ala Pro GlyThr Ile Met 325 330 335 Ala His Ala Asp Leu Trp Thr Phe Thr Glu Asn PheIle Val Met Pro 340 345 350 His Arg Ala Phe Gly Gly Ile Lys Asp Leu GlyGln Pro Gly Tyr Asn 355 360 365 Lys Ile Asn Asp Met Ile Asn Asn Ile ThrThr Lys Ser Val Phe Ile 370 375 380 9 4881 DNA feline herpesvirus 1 9ttcgactcta caatcttgta gattttgggt tgtaggtggt gtgttgtcga ttggaggtcc 60accaacagtg acggtctcta ccggccatcc ccattccccg agaatcgtca aaataggtct 120gatatcgtcc atctacgtta ggttggagaa gcaccataaa cgacgtagtt ttgctgtggg 180ttttaagtat accgaaggcc tccttatcgc gtaagttgtt ttcgtcatat acttcactca 240ttcaacactc caatcgatgg ctcccttacc cctctttaca agaaactcat ttgccattct 300aaaatgcgta cactcgctaa atgggatacg tgacagtgga gatggatggg tatatgtcag 360tatcaaatgc cgatgtggat ctgggttaca agctttttgg gcgtgggatc cccataacat 420aaataccagc ccatcagaaa tcgatgccag atgatctaat atagccttaa ctaatttttc 480ccaaccaatc attctatgag atccaggact cccccgtcta actgtgagtg tagtgttaat 540taataatact cctctttcca cccatttaag aagattacca tgcttcccga ttcggatatc 600ggggtaattc ttccgtagtg ccgcgtagat gtttctgaga ctcggaggaa tggttacgtt 660ctcatgaacg ctaaacgcca ggccattggc ttgaccgggt ccgtggtatg gatcttgtcc 720caagatgact actttaatgt ccatgggttg tatcgcacgt gtccatgaaa atatatcttg 780tattttaggt aggacctctt catagcgtag acgacgttcg tattccagta aaatgcgttg 840ggtatacggt ttcttaaatt caggttcgag aatagatgcc catgaatgtg atatattaaa 900ctttcgttgg atagtcatcc acgaaatcgg tttctctata ttggaaacca cttgtacatc 960tggatctaaa attactccag atgggagacc ggtgggtctc aatttttttc ttggtggctc 1020tgtagatgta gtatctgatg gagcattggt agtgcaatag gtatggtcga gagctacaac 1080agctgaatta tgattcacaa gctcattcat agttgacata gatataaggt tagtatatct 1140cagcgatata tactaacctt ttttgtttgt aggttaagga agctttatac tatcttgtag 1200agatggagag tcacgtggag gcggtggaag agtatctgca tccggggcaa gttgatctcg 1260tcggaaagct gtgtccagaa catcagtgaa tctggatttt aagtgtattg ggacggacat 1320ccgtctgaca tcttcagcca atccctgtag tgcaacaaag ggattaaccc aatatgctcg 1380tttatcccct ttaaaccaca gtattacctc cggtgggtta caatctgctc gcacgataat 1440tccagccaaa tcttctttga tattttcatc acctagtcca tataattttt tttctactcg 1500gtgatcacgg catggtccta aaagaattac atcaatccca tcgtattgga cttgctcctc 1560cgaggcacca aaatctggaa tataaatctt caagagtaat aaaacgaaga cccacatcgt 1620ggaaagacca gcggtcgata tacctgtgaa aatcacacaa cctaatatgt agaacggtct 1680caaaaaccaa ctaacccaac ccaatagtga gtgccattta tatagtatgg tatacaccct 1740gaaatacagt caaatgtcat atacatttat gtgattcaaa tatttattat gcccgtctat 1800tagacagttt acagtattcg attatgtagt actcctaata tccggtagat agcgtatttg 1860tctgcaaaac attcttctgt agtggactct ggtaataact cctatcgatc tataaacaaa 1920aagccatttg tttaagccat taagggggaa cttaagattc tggaaaaagc tattccagcc 1980attaaatggg aacacagagt ttggaaacag cacactacat ccttttagta agaattcatg 2040aataaaatat aattccacat ttattatata ggtccgtata gctgcgtgcg ggtgggagca 2100tctgaggcta ttacatcccg gggcaagatg tacgaggaca tcgtgataaa gataagtaac 2160aatttcctac cgggggttta tatccagaat attaaatgct gggggcccac caatctcggc 2220ggtctttatt ttgtataaac tgacaacact gagtttcatg tgtattacta tctttattaa 2280tgatatatcc aacaataatt acattgttct actacaatct ggtattcggt atatgtacga 2340tcggcccaga taccctccta cgacccctgc gggttgtctg taaccgtgta tgagaaatcg 2400cagtggtcga agagtctcgt tgagtcatat ttgacgtcga tggggttgat actaaattaa 2460aggtttctgg gctccccctc aatctagcag acctccgctc gttccatcga gacgggggtc 2520gtttgcgctt tctatcggtt ggggttacgc ttatgggagt attatgacta ccagaatcgg 2580tgttgggagg tccatcggca tcatgaacac ctctgatatc atcctcattt ttttgatacc 2640tggatgaaac tgtagtattt actataggat cggctctgcc gtcatcatag gaaactgcag 2700tattttctgt agtattggtt ctgtcgttat ttgcgccatc tagagctatt acagtagtac 2760tgttttcatt agaactttct gtgtcatgag ccagagtaag gtctaaaggt tccatattgg 2820tctccggata gttctgttgt atcgattcag ccggagccgc gaaattggta gccgatgacc 2880cgtgtggaag tgtagataac tcaggctgtg tatttagtga atgacgtgtc tcatcatcta 2940tccctgtagt gataccaaca gataagtctg tgccaattgt ggtctctgta tcgtcagaat 3000catcggtatc atcatatgca gtgtcctcgg ggtctgtgag atctgctgtg gcgagactag 3060acgattcatc gtcggaggag gtatcgtctt ctgtatcatc taggaattgg accccggttg 3120aaacgcgtaa agtacgggcc gtagaaggaa atacgcactg tgatgcttcg gcgagcaacc 3180gtatcacgaa gagatccgtc tgtgtgtgga gcaggggtct gagaaggcga gtaagcgcag 3240cgctattgaa tgcatgctcg cacagctcat ccataacaat accacacatg acactggctg 3300ttgtctcatc gttgtaataa ttcaaaagcc agctgttcat ccacgttact agagggcgca 3360gatgtggggc tccggcaccc ccgtctgggg tcaggggcac aaatcccagt cctgtagatg 3420tagaatcccg tggtcttgtt gaagttggat tacctgcccc atcgctgggt cgtccagagg 3480tatcaaccgt tgttgaattt tcaagacgaa cactgttcag agatatatta gcccatcgtc 3540gggcttcagt ccatataaac ggatcccaat cgagatactc ctcctcctct ctatgtgggc 3600tatatagttt cgtttcttca aagattttat cgtcctctat agagtggatg agcgatgtca 3660cactagtttt acagagggga catttgttat ttaatcctac ccacctactg agacaggaat 3720agcaaaattt atgcaaacat ggcatggtga aagttaaatc attcatggga tcaaggcata 3780ttggacagtg gtctcccata tcagccatgt cgaggtcctc tacccagacc aaaaagagtt 3840atgtcaggtc cttacaaatc aatcagaccc aagtcggact cacgacttta atatataccc 3900ataacaggaa gtgagctctt gggatatata tgttgtcagt gttcatcaaa attctcattc 3960ggtagacaca tgtgataggg tttcgtttct agaaatacct cccatactaa aatgtaatgt 4020tctaatatat cgttctgtga tctatgtaaa taatagcagc gcatcgagta tctgctaggc 4080gggctttctt ctggatctta tccatcggaa atccacaccg gcccaaagct tcaagttact 4140ttcactttca gtgatttata aactacttag aatcccataa gacctcttac ttgtattgat 4200tttatgatat cttggggctt tcccctgact aaaaattcac cgggtcttgt ttcatgttac 4260cacagtccaa aacatttaat ataatagtta tttttatctc acatgcaaaa aatataatag 4320gtggataagt atcaatgttc ttttccaaaa aaatcacatt cccattcgta tctcggaatg 4380gctacctgca tgtctggcct tagtactaaa tcgcatgttt ctatacgatg tatctgattt 4440gcatgctcgt gtatcatgta tcggtcatgg ggatcccact acgttccact aaaaaccatt 4500tctagacatc ccgaagttcg gatggaatgt tggttacttc ccccactccg gccgccgcgg 4560gagatctcgg cccccctagc cacttttccg agggggtcca aaaagggggc ggggtctttt 4620tttggggggc gtggctaggg ggcctcccct tccttattag gcccctcccc ttccttatta 4680ggcccctccc cttccttatt aggcccctcc ccttccttat taggcccctc cccttcctta 4740ttaggcccct ccccttcctt attaggcccc tccccttcct tattaggccc ctccccttcc 4800ttattaggcc cctccccttc cttattaagc ccctcccctt ccttattagg cccctcccct 4860tccttattaa gcccctcccc t 4881 10 293 PRT feline herpesvirus 1 10 Met SerThr Met Asn Glu Leu Val Asn His Asn Ser Ala Val Val Ala 1 5 10 15 LeuAsp His Thr Tyr Cys Thr Thr Asn Ala Pro Ser Asp Thr Thr Ser 20 25 30 ThrGlu Pro Pro Arg Lys Lys Leu Arg Pro Thr Gly Leu Pro Ser Gly 35 40 45 ValIle Leu Asp Pro Asp Val Gln Val Val Ser Asn Ile Glu Lys Pro 50 55 60 IleSer Trp Met Thr Ile Gln Arg Lys Phe Asn Ile Ser His Ser Trp 65 70 75 80Ala Ser Ile Leu Glu Pro Glu Phe Lys Lys Pro Tyr Thr Gln Arg Ile 85 90 95Leu Leu Glu Tyr Glu Arg Arg Leu Arg Tyr Glu Glu Val Leu Pro Lys 100 105110 Ile Gln Asp Ile Phe Ser Trp Thr Arg Ala Ile Gln Pro Met Asp Ile 115120 125 Lys Val Val Ile Leu Gly Gln Asp Pro Tyr His Gly Pro Gly Gln Ala130 135 140 Asn Gly Leu Ala Phe Ser Val His Glu Asn Val Thr Ile Pro ProSer 145 150 155 160 Leu Arg Asn Ile Tyr Ala Ala Leu Arg Lys Asn Tyr ProAsp Ile Arg 165 170 175 Ile Gly Lys His Gly Asn Leu Leu Lys Trp Val GluArg Gly Val Leu 180 185 190 Leu Ile Asn Thr Thr Leu Thr Val Arg Arg GlySer Pro Gly Ser His 195 200 205 Arg Met Ile Gly Trp Glu Lys Leu Val LysAla Ile Leu Asp His Leu 210 215 220 Ala Ser Ile Ser Asp Gly Leu Val PheMet Leu Trp Gly Ser His Ala 225 230 235 240 Gln Lys Ala Cys Asn Pro AspPro His Arg His Leu Ile Leu Thr Tyr 245 250 255 Thr His Pro Ser Pro LeuSer Arg Ile Pro Phe Ser Glu Cys Thr His 260 265 270 Phe Arg Met Ala AsnGlu Phe Leu Val Lys Arg Gly Lys Gly Ala Ile 275 280 285 Asp Trp Ser ValGlu 290 11 147 PRT feline herpesvirus 1 11 Met Trp Val Phe Val Leu LeuLeu Leu Lys Ile Tyr Ile Pro Asp Phe 1 5 10 15 Gly Ala Ser Glu Glu GlnVal Gln Tyr Asp Gly Ile Asp Val Ile Leu 20 25 30 Leu Gly Pro Cys Arg AspHis Arg Val Glu Lys Lys Leu Tyr Gly Leu 35 40 45 Gly Asp Glu Asn Ile LysGlu Asp Leu Ala Gly Ile Ile Val Arg Ala 50 55 60 Asp Cys Asn Pro Pro GluVal Ile Leu Trp Phe Lys Gly Asp Lys Arg 65 70 75 80 Ala Tyr Trp Val AsnPro Phe Val Ala Leu Gln Gly Leu Ala Glu Asp 85 90 95 Val Arg Arg Met SerVal Pro Ile His Leu Lys Ser Arg Phe Thr Asp 100 105 110 Val Leu Asp ThrAla Phe Arg Arg Asp Gln Leu Ala Pro Asp Ala Asp 115 120 125 Thr Leu ProPro Pro Pro Arg Asp Ser Pro Ser Leu Gln Asp Ser Ile 130 135 140 Lys LeuPro 145 12 498 PRT feline herpesvirus 1 12 Met Ala Asp Met Gly Asp HisCys Pro Ile Cys Leu Asp Pro Met Asn 1 5 10 15 Asp Leu Thr Phe Thr MetPro Cys Leu His Lys Phe Cys Tyr Ser Cys 20 25 30 Leu Ser Arg Trp Val GlyLeu Asn Asn Lys Cys Pro Leu Cys Lys Thr 35 40 45 Ser Val Thr Ser Leu IleHis Ser Ile Glu Asp Asp Lys Ile Phe Glu 50 55 60 Glu Thr Lys Leu Tyr SerPro His Arg Glu Glu Glu Glu Tyr Leu Asp 65 70 75 80 Trp Asp Pro Phe IleTrp Thr Glu Ala Arg Arg Trp Ala Asn Ile Ser 85 90 95 Leu Asn Ser Val ArgLeu Glu Asn Ser Thr Thr Val Asp Thr Ser Gly 100 105 110 Arg Pro Ser AspGly Ala Gly Asn Pro Thr Ser Thr Arg Pro Arg Asp 115 120 125 Ser Thr SerThr Gly Leu Gly Phe Val Pro Leu Thr Pro Asp Gly Gly 130 135 140 Ala GlyAla Pro His Leu Arg Pro Leu Val Thr Trp Met Asn Ser Trp 145 150 155 160Leu Leu Asn Tyr Tyr Asn Asp Glu Thr Thr Ala Ser Val Met Cys Gly 165 170175 Ile Val Met Asp Glu Leu Cys Glu His Ala Phe Asn Ser Ala Ala Leu 180185 190 Thr Arg Leu Leu Arg Pro Leu Leu His Thr Gln Thr Asp Leu Phe Val195 200 205 Ile Arg Leu Leu Ala Glu Ala Ser Gln Cys Val Phe Pro Ser ThrAla 210 215 220 Arg Thr Leu Arg Val Ser Thr Gly Val Gln Phe Leu Asp AspThr Glu 225 230 235 240 Asp Asp Thr Ser Ser Asp Asp Glu Ser Ser Ser LeuAla Thr Ala Asp 245 250 255 Leu Thr Asp Pro Glu Asp Thr Ala Tyr Asp AspThr Asp Asp Ser Asp 260 265 270 Asp Thr Glu Thr Thr Ile Gly Thr Asp LeuSer Val Gly Ile Thr Thr 275 280 285 Gly Ile Asp Asp Glu Thr Arg His SerLeu Asn Thr Gln Pro Glu Leu 290 295 300 Ser Thr Leu Pro His Gly Ser SerAla Thr Asn Phe Ala Ala Pro Ala 305 310 315 320 Glu Ser Ile Gln Gln AsnTyr Pro Glu Thr Asn Met Glu Pro Leu Asp 325 330 335 Leu Thr Leu Ala HisAsp Thr Glu Ser Ser Asn Glu Asn Ser Thr Thr 340 345 350 Val Ile Ala LeuAsp Gly Ala Asn Asn Asp Arg Thr Asn Thr Thr Glu 355 360 365 Asn Thr AlaVal Ser Tyr Asp Asp Gly Arg Ala Asp Pro Ile Val Asn 370 375 380 Thr ThrVal Ser Ser Arg Tyr Gln Lys Asn Glu Asp Asp Ile Arg Gly 385 390 395 400Val His Asp Ala Asp Gly Pro Pro Asn Thr Asp Ser Gly Ser His Asn 405 410415 Thr Pro Ile Ser Val Thr Pro Thr Asp Arg Lys Arg Lys Arg Pro Pro 420425 430 Ser Arg Trp Asn Glu Arg Arg Ser Ala Arg Leu Arg Gly Ser Pro Glu435 440 445 Thr Phe Asn Leu Val Ser Thr Pro Ser Thr Ser Asn Met Thr GlnArg 450 455 460 Asp Ser Ser Thr Thr Ala Ile Ser His Thr Arg Leu Gln ThrThr Arg 465 470 475 480 Arg Gly Arg Arg Arg Val Ser Gly Pro Ile Val HisIle Pro Asn Thr 485 490 495 Arg Leu 13 33 DNA feline herpesvirus 13gaatacacgg aattaattcg atgccacaat cgc 33 14 26 DNA feline herpesvirus 14aaaggctgct gcctcgacgt ctgggg 26 15 34 DNA feline herpesvirus 15gcctggtgtc cgtcgaggca gctttcctag ggag 34 16 43 DNA feline herpesvirus 16tccgtactat ctattgtcga aattcgagct cgcccgggga tcc 43 17 33 DNA felineherpesvirus 17 gaatacacgg aattaattcg atgccacaat cgc 33 18 36 DNA felineherpesvirus 18 tgacactcca ttggcatcga cggacaccag gcgccg 36 19 37 DNAfeline herpesvirus 19 gcgccccaga cgtcgatgaa acaaacctag ccataga 37 20 43DNA feline herpesvirus 20 tccgtactat ctattgtcga aattcgagct cgcccggggatcc 43 21 31 DNA Artificial Sequence misc_feature ()..() PCR andsynthetic primer 21 gcaactgcag caacaatgaa ttcggatccc g 31 22 33 DNAArtificial Sequence misc_feature ()..() PCR and synthetic primer 22cgttctgcag cctctagctt attctagatc ttt 33 23 74 DNA feline herpesvirus 23gttcacagta tctactgtgc acctaccccg gggatcctct agagtcgact gcagcaacaa 60tcaattcgga tccc 74

What is claimed is:
 1. A recombinant feline herpesvirus comprising a foreign DNA inserted into a feline herpesvirus genome, wherein the foreign DNA is inserted into a region of the genome which corresponds to a 3.0 kb EcoRI-SalI fragment within a SalI A fragment of the feline herpesvirus genome and is capable of being expressed in a host cell into which the virus is introduced.
 2. The recombinant feline herpesvirus of claim 1, wherein the foreign DNA is inserted within a UL25 gene within the region which corresponds to the 3.0 kb EcoRI-SalI fragment.
 3. The recombinant feline herpesvirus of claim 1, wherein the foreign DNA is inserted within a NdeI site within the region which corresponds to the 3.0 kb EcoRI-SalI fragment.
 4. The recombinant feline herpesvirus of claim 1, further comprising a second foreign DNA inserted within a non-essential region of the feline herpesvirus genome.
 5. The recombinant feline herpesvirus of claim 4, wherein the second foreign DNA is inserted within a unique short region of the feline herpesvirus genome.
 6. The recombinant feline herpesvirus of claim 4, wherein the second foreign DNA is inserted within a glycoprotein E gene within the unique short region of the feline herpesvirus genome.
 7. The recombinant feline herpesvirus of claim 4, the second foreign DNA is inserted within a glycoprotein I gene within the unique short region of the feline herpesvirus genome.
 8. The recombinant feline herpesvirus of claim 4, the second foreign DNA is inserted within a glycoprotein G gene within the unique short region of the feline herpesvirus genome.
 9. The recombinant feline herpesvirus of claim 1, further comprising a deletion in a non-essential region of the feline herpesvirus genome.
 10. The recombinant feline herpesvirus of claim 9, wherein the deletion is in the unique short region.
 11. The recombinant feline herpesvirus of claim 1, wherein the foreign DNA encodes a screeneable marker.
 12. The recombinant feline herpesvirus of claim 11, wherein the screenable marker is E. coli beta-galactosidase.
 13. The recombinant feline herpesvirus of claim 11, wherein the screenable marker is E. coli beta-glucouronidase.
 14. The recombinant feline herpesvirus of claim 1, wherein the foreign DNA encodes a polypeptide.
 15. The recombinant feline herpesvirus of claim 1, wherein the- foreign DNA encodes Marek's Disease Virus glycoprotein A, Marek's Disease Virus glycoprotein B or Marek's Disease Virus glycoprotein D.
 16. The recombinant feline herpesvirus of claim 1, wherein the foreign DNA encodes Newcastle Disease Virus fusion protein or Newcastle Disease Virus hemagglutinin-neuraminidase.
 17. The recombinant feline herpesvirus of claim 1, wherein the foreign DNA encodes Infectious Laryngotracheitis Virus glycoprotein B Infectious Laryngotracheitis Virus glycoprotein I or Infectious Laryngotracheitis Virus glycoprotein D.
 18. The recombinant feline herpesvirus of claim 1, wherein the foreign DNA encodes Infectious Bronchitis Virus spike protein or Infectious Bronchitis Virus matrix protein.
 19. The recombinant feline herpesvirus of claim 1, wherein the foreign DNA encodes Infectious Bursal Disease virus VP2, Infectious Bursal Disease virus VP3, or Infectious Bursal Disease virus VP4.
 20. The recombinant feline herpesvirus of claim 1, wherein the foreign DNA is selected from the group consisting of: Feline Leukemic virus envelope gene, Hepatitis B core antigen gene, Pseudorabies virus glycoprotein C gene, Dirofilaria immitis 22 kD gene, Dirofilaria immitis p39 gene, Feline Immunodeficiency virus gag gene, Feline Immunodeficiency virus pol gene, and Feline Immunodeficiency virus env gene.
 21. The recombinant feline herpesvirus of claim 1, wherein the foreign DNA encodes a cytokine.
 22. The recombinant feline herpesvirus of claim 1, wherein the foreign DNA is under the control of an endogenous upstream promoter.
 23. The recombinant feline herpesvirus of claim 1, wherein the foreign DNA is under the control of a heterologous upstream promoter.
 24. The recombinant feline herpesvirus of claim 23, wherein the promoter is selected from the group consisting of: Pseudorabies virus glycoprotein X promoter, Herpes Simplex Virus-1 alpha 4 promoter, Human Cytomegalovirus immediate early promoter, Marek's Disease Virus glycoprotein A promoter, Marek's Disease Virus glycoprotein B promoter, Marek's Disease Virus glycoprotein D promoter, Infectious Laryngotracheitis Virus glycoprotein B promoter, Infectious Laryngotracheitis Virus glycoprotein D promoter, and Bovine Herpesvirus-1.1 VP8 promoter and chicken anemia virus promoter.
 25. The recombinant feline herpesvirus of claim 22, wherein the promoter is feline herpesvirus glycoprotein E promoter. 